Methods and compositions for inhibition of polymerase

ABSTRACT

The present invention is directed to methods and compositions for inhibition of viral nucleic acid polymerases, such as RNA and DNA polymerases, and methods and compositions that are useful for treating viral infections in subjects. The methods comprise administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. The composition or method may optionally comprise one or more additional anti-viral agents.

This application claims priority to U.S. Provisional Application No.61/393,522, filed on Oct. 15, 2010 and U.S. Provisional Application No.61/492,054, filed on Jun. 1, 2011, both of which are incorporated hereinby reference.

The disclosures of all patents, patent applications and publicationscited herein are hereby incorporated by reference in their entirety.

BACKGROUND

Viral diseases are responsible for both global pandemics and yearlyseasonal epidemics such as influenza. Outbreaks may be characterized bypotentiated virulence and may occur suddenly, resulting in seriousmortalities. Importantly, viral diseases are not limited to humans. Forexample, influenza also affects livestock and birds, which may havesignificant impact on food supply in addition to increasing the risk oftransmission to humans. Exemplary conditions related to viral infectioninclude, for example, influenza, small pox, encephalitis, West Niledisease, yellow fever, Dengue fever, hepatitis, human immunodeficiency,polio, and Coxsackie.

The genome of the influenza A virus has an RNA-dependent RNA polymerase,which is a heterotrimeric complex of three subunits (PA, PB1 and PB2).The RNA polymerase catalyzes viral RNA transcription and replication.Because transcription and replication of the virus depends on theactivity of RNA polymerase, this enzyme has become of interest as atarget for development of new anti-viral compounds, especially in thewake of the recent emergence of drug resistant viruses.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for inhibition of viralnucleic acid polymerases, and methods and compositions that are usefulfor treating, suppressing and/or preventing viral infections insubjects. The methods comprise administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt or hydrate thereof, or a compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt or hydrate thereof, and a pharmaceutically acceptable carrier. Thecomposition or method may optionally comprise one or more additionalanti-viral agents. The methods and compositions are useful for treating,suppressing and/or preventing viral infections in subjects that mayarise from infection by one or more type of virus. Thus, the methods andcompositions are useful for broad spectrum anti-viral treatment,suppression, and/or prevention.

The present invention is based, in part, on certain discoveries whichare described more fully in the Examples section of the presentapplication. For example, the present invention is based, in part, onthe discovery that levels of viral titer in cells were markedly reducedupon treatment with a compound of formula I. Thus, the present inventionalso provides methods for reducing viral titer in a bodily fluid or cellcomprising contacting said fluid or cell with a compound of formula I.The present invention is further based, in part, on the discovery thatlevels of viral titer in cells for several viruses were markedly reducedupon treatment with a compound of formula I, indicating broad spectrumantiviral activity for the compound of formula I against a variety ofviral strains. Thus, the present invention also provides methods forreducing viral titer for several types, subtypes and/or strains ofviruses in a bodily fluid or cell comprised of contacting said fluid orcell with a compound of formula I.

In some embodiments, the present invention provides a method forinhibiting a viral RNA or DNA polymerase comprising contacting thepolymerase with an effective inhibitory amount of the compound offormula I, or a pharmaceutically acceptable salt, solvate, or hydratethereof.

In some embodiments, the method is performed in vivo.

In some embodiments, the present invention provides a method fortreating a subject suffering from an RNA viral infection which comprisesadministering to said subject a therapeutically effective amount of acompound of formula I, or pharmaceutically acceptable salt thereof.

In some embodiments, the bodily fluid is blood. In some embodiments, thebodily fluid is plasma. In some embodiments, the bodily fluid is bloodserum.

In some embodiments, the subject is a mammal. In some embodiments thesubject is a human. In some embodiments, the subject is avian. In someembodiments, the subject is a swine or pig.

These and other embodiments of the invention are further described inthe following sections of the application, including the DetailedDescription, Examples, and Claims.

Still other objects and advantages of the invention will become apparentto those of skill in the art from the disclosure herein, which is simplyillustrative and not restrictive. Thus, other embodiments will berecognized by the skilled artisan without departing from the spirit andscope of the invention.

SUMMARY OF FIGURES

FIG. 1 shows the phosphorylation of compound 1 in human hepatocellularcarcinoma (Huh-7) cells.

FIG. 2 shows phosphorylation of ³H adenosine in Huh-7 cells.

FIG. 3 shows phosphorylation of ³H compound 1 in Huh-7 cells.

FIG. 4 shows total RNA and genomic DNA incorporation of ³H compound 1and ³H adenosine in Huh-7 cells after 24 h.

FIG. 5 shows the combination effects of compound 1 and peramivir (aneuraminidase inhibitor) on influenza in vitro.

FIG. 6 shows the effect of compound 1 (intramuscular) on weight loss inmice infected with H3N2 A/Victoria/3/75 influenza virus.

FIG. 7 shows the effect of compound 1 (oral) on weight loss in miceinfected with H3N2 A/Victoria/3/75 influenza virus.

FIG. 8 shows the effect of compound 1 (intraperitoneal, intramuscularand oral) on survival of mice infected with Ebola virus.

FIG. 9 shows the effect of compound 1 (intraperitoneal, intramuscularand oral) on weight loss in mice infected with Ebola virus.

FIG. 10 shows the effect of compound 1 (intramuscular and oral) onsurvival of mice infected with Ebola virus.

FIG. 11 shows the effect of compound 1 (intramuscular and oral) onweight loss in mice infected with Ebola virus.

FIG. 12 shows the effect of compound 1 on survival of hamsters infectedwith Yellow Fever virus.

FIG. 13 shows the effect of compound 1 on weight loss in hamstersinfected with Yellow Fever virus.

FIG. 14 shows the oral pharmacokinetic curve of compound 1 dosed at 10mg/kg as measured in rats.

DETAILED DESCRIPTION

The invention provides methods and compositions for inhibition of viralnucleic acid polymerases, such as RNA and DNA polymerases, and methodsand compositions that are useful for treating viral infections insubjects. The methods comprise administering to the subject atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt or hydrate thereof, or a compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt or hydrate thereof, and a pharmaceutically acceptable carrier. Thecomposition or method may optionally comprise one or more additionalanti-viral agents. The methods and compositions are useful for treating,suppressing and/or preventing viral infections in subjects that mayarise from infection with one or more type of virus. Thus, the methodsand compositions are useful for broad spectrum anti-viral treatment,suppression, and/or prevention.

In particular, the present invention relates to methods of treatment,suppression or and/or prevention of diseases or conditions relating toviral infection comprising administration of a compound of formula I, orpharmaceutically acceptable salt or hydrate thereof.

The compounds of formula (I) are as follows:

wherein A is OH or NH₂, and B is H or NH_(2.)

Thus, in some embodiments of the compound of formula (I), A is NH₂.

In some embodiments of the compound of formula (I), B is NH₂.

In some embodiments of the compound of formula (I), A is OH.

In yet some embodiments of the compound of formula (I), B is H.

In still some embodiments of the compound of formula (I), A is NH₂ and Bis H.

In still some embodiments of the compound of formula (I), A is OH and Bis NH₂.

In still some embodiments of the compound of formula (I), A is NH₂ and Bis NH₂.

In still some embodiments of the compound of formula (I), A is OH and Bis H.

The present invention is based, in part, on certain discoveries whichare described more fully in the Examples section of the presentapplication. For example, the present invention is based, in part, onthe discovery that levels of viral titer in cells were markedly reducedupon treatment with a compound of formula I. Thus, in some embodiments,the present invention provides methods for reducing viral titer in abodily fluid or cell comprised of contacting said fluid or cell with acompound of formula I. The present invention is further based, in part,on the discovery that levels of viral titer in cells for several viruseswere markedly reduced upon treatment with a compound of formula I, thusindicating broad spectrum antiviral activity for the compound of formulaI against a variety of viral strains. Thus, the present invention alsoprovides methods for reducing viral titer for several types, subtypesand/or strains of viruses in a bodily fluid or cell comprised ofcontacting said fluid or cell with a compound of formula I.

The compounds of the present invention are prepared in different forms,such as salts, hydrates, solvates, or complexes, and the inventionincludes compositions and methods encompassing all variant forms of thecompounds. In some embodiments, the compounds are prepared as hydratesof salts.

Abbreviations and Definitions

The abbreviation “PNP” refers to purine nucleoside phosphorylase.

The term “compound(s) of the invention” as used herein means a compoundof formula I, and may include salts, tautomeric forms, hydrates and/orsolvates thereof. Compounds of formula I may also include solvates orhydrates of salts thereof.

The term “solvate” as used herein means a compound of formula I, or apharmaceutically acceptable salt thereof, wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent is physiologically tolerable at the dosage administered.Examples of suitable solvents are ethanol, water and the like. Whenwater is the solvent, the molecule is referred to as a “hydrate.”

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or pharmaceutically acceptable salts, orhydrates thereof, with other chemical components, such asphysiologically acceptable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of a compoundto an organism.

The term “pharmaceutically acceptable salt” is intended to include saltsderived from inorganic or organic acids including, for examplehydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric,formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic,salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic,trifluroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids.Pharmaceutically acceptable salt forms may also include forms whereinthe ratio of molecules comprising the salt is not 1:1. For example, thesalt may comprise more than one inorganic or organic acid molecule permolecule of base, such as two hydrochloric acid molecules per moleculeof compound of formula (I). As another example, the salt may compriseless than one inorganic or organic acid molecule per molecule of base,such as two molecules of compound of formula (I) per molecule oftartaric acid. Salts may also exist as solvates or hydrates.

The term “acid” contemplates all pharmaceutically acceptable inorganicor organic acids. Inorganic acids include mineral acids such ashydrohalic acids, such as hydrobromic and hydrochloric acids, sulfuricacids, phosphoric acids and nitric acids. Organic acids include allpharmaceutically acceptable aliphatic, alicyclic and aromatic carboxylicacids, dicarboxylic acids, tricarboxylic acids, and fatty acids.Preferred acids are straight chain or branched, saturated or unsaturatedC1-C20 aliphatic carboxylic acids, which are optionally substituted byhalogen or by hydroxyl groups, or C6-C12 aromatic carboxylic acids.Examples of such acids are carbonic acid, formic acid, fumaric acid,acetic acid, propionic acid, isopropionic acid, valeric acid,alpha-hydroxy acids, such as glycolic acid and lactic acid, chloroaceticacid, benzoic acid, methane sulfonic acid, and salicylic acid. Examplesof dicarboxylic acids include oxalic acid, malic acid, succinic acid,tataric acid and maleic acid. An example of a tricarboxylic acid iscitric acid. Fatty acids include all pharmaceutically acceptablesaturated or unsaturated aliphatic or aromatic carboxylic acids having 4to 24 carbon atoms. Examples include butyric acid, isobutyric acid,sec-butyric acid, lauric acid, palmitic acid, stearic acid, oleic acid,linoleic acid, linolenic acid, and phenylsteric acid. Other acidsinclude gluconic acid, glycoheptonic acid and lactobionic acid.

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

An “effective amount”, “sufficient amount” or “therapeutically effectiveamount” as used herein is an amount of a compound that is sufficient toeffect beneficial or desired results, including clinical results. Assuch, the effective amount may be sufficient, for example, to reduce orameliorate the severity and/or duration of the viral infection, or oneor more symptoms thereof, prevent the advancement of the viralinfection, prevent the recurrence, development, or onset of one or moresymptoms associated with the viral infection, prevent or reduce thereplication or multiplication of a virus, prevent or reduce theproduction and/or release of a viral particle, enhance or otherwiseimprove the prophylactic or therapeutic effect(s) of another therapy. Aneffective amount also includes the amount of the compound of formula Ithat avoids or substantially attenuates undesirable side effects.

As used herein and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results may include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminution of extent of disease, a stabilized (i.e., notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Non-limiting examples of suchpharmaceutical carriers include liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical carriers may also be saline, gum acacia, gelatin,starch paste, talc, keratin, colloidal silica, urea, and the like. Inaddition, auxiliary, stabilizing, thickening, lubricating and coloringagents may be used. Other examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin;herein incorporated by reference in its entirety.

The terms “animal,” “subject” and “patient” as used herein include allmembers of the animal kingdom including, but not limited to, mammals,animals (e.g., cats, dogs, horses, swine, etc.) and humans.

Description

The present invention provides methods and compositions for inhibitionof viral nucleic acid polymerases, such as DNA and/or RNA viralpolymerases, and methods and compositions that are useful for treatingviral infections in subjects. The methods comprise administering to thesubject a therapeutically effective amount of a compound of formula I,or a pharmaceutically acceptable salt thereof, or a compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt or hydrate thereof, and a pharmaceutically acceptable carrier. Thecomposition or method may optionally comprise one or more additionalanti-viral agents. The methods and compositions are useful for treating,suppressing and/or preventing viral infections in subjects that mayarise from infection by one or more family, genus, subtype, serotype, orstrain of virus.

The compounds of formula I are 9-deazaadenine derivatives generallyknown as immucillins, the syntheses of which are described, for example,in WO 03/80620, and by Evans et al., in Tetrahedron 2000, 56, 3053 andJ. Org. Chem. 2001, 66(17), 5723 (each of which herein incorporated byreference in its entirety). Syntheses of similar structures arediscussed, for example, in U.S. Pat. Nos. 5,985,848; 6,066,722;6,228,741 and PCT publications WO 2003/080620 and 2008/030119 (each ofwhich herein incorporated by reference in its entirety). Immucillinderivatives have been studied as PNP inhibitors (See, Kicska et al., J.Biol. Chem. 2002, 277, 3219-3225, and Kicska et al., J. Biol. Chem.2002, 277, 3226-3231; each of which herein incorporated by reference inits entirety). Some immucillins have also been studied as5′-methylthioadenosine phosphorylase (MTAP) or 5′-methylthioadenosinenucleosidase (MTAN) inhibitors. Such mechanisms have been implicated inthe treatment of cancer and bacterial infections (See, WO 03/080620,herein incorporated by reference in its entirety).

The compounds of formula I may exhibit tautomeric properties. Thus, thepresent invention also encompasses tautomeric forms of compounds offormula I, and mixtures thereof. It will further be appreciated thatsome compounds exist as pharmaceutically acceptable salts, solvates,and/or hydrates, each of which are also within the embodiments of theinvention.

In some embodiments, the compound of formula I exists as apharmaceutically acceptable salt. In some embodiments, the salt form isabout a 1:1 ratio of acid and compound of formula I. In someembodiments, the salt form is greater than about a 1:1 ratio of acid andcompound of formula I. In some embodiments, the salt form is about a 2:1ratio of acid and compound of formula I. In some embodiments, the saltform exists as a hydrate.

In some embodiments, the compound of formula I exists as a hydrate orsolvate.

The compounds of the disclosure therefore are useful in treating and/orpreventing viral infections in a host or subject. The methods of theinvention may be used in treating and/or preventing disease states orconditions caused by and/or related to such viral infections. Examplesof such viral infections include, but are not limited to, adenovirus,rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola,Coxsackie, Rhino, West Nile, small pox, encephalitis, yellow fever,Dengue fever, influenza (including human, avian, and swine), lassa,lymphocytic choriomeningitis, junin, machuppo, guanarito, hantavirus,Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo,Marburg, Japanese Encephalitis, Kyasanur Forest, Venezuelan equineencephalitis, Eastern equine encephalitis, Western equine encephalitis,severe acute respiratory syndrome (SARS), parainfluenza, respiratorysyncytial, Punta Toro, Tacaribe and pachindae.

In a some embodiments, the compounds of the invention are used to treator prevent a viral infection associated with a virus. In someembodiments, the viral infection comprises infection with one or moretype of virus. In some embodiments, the viral infection comprisesinfection by one or more viruses selected from the group consisting oforthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae,flaviviridae, filoviridae, togaviridae, picornaviridae, andcoronaviridae viruses. In some embodiments, the viral infectioncomprises infection by one or more viruses selected from the groupconsisting of adenovirus, rhinovirus, hepatitis, immunodeficiency virus,polio, measles, Ebola, Coxsackie, Rhino, West Nile, small pox,encephalitis, yellow fever, Dengue fever, influenza (including human,avian, and swine), lassa, lymphocytic choriomeningitis, junin, machuppo,guanarito, hantavirus, Rift Valley Fever, La Crosse, Californiaencephalitis, Crimean-Congo, Marburg, Japanese Encephalitis, KyasanurForest, Venezuelan equine encephalitis, Eastern equine encephalitis,Western equine encephalitis, severe acute respiratory syndrome (SARS),parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindaeviruses.

In some embodiments, the viral infection comprises infection by one ormore viruses selected from the group consisting of adenovirus, Denguefever, influenza A and influenza B (including human, avian, and swine),junin, measles, parainfluenza, Pichinde, punta toro, respiratorysyncytial, rhinovirus, Rift Valley Fever, severe acute respiratorysyndrome (SARS), Tacaribe, Venezuelan equine encephalitis, West Nile andyellow fever viruses.

In some embodiments, the virus is Ebola, Marburg, yellow fever,influenza A or influenza B. In some embodiments, the virus is Ebola. Insome embodiments, the virus is Marburg. In some embodiments, the virusis yellow fever. In some embodiments, the virus is influenza A orinfluenza B.

In some embodiments, the virus is West Nile or Dengue fever. In someembodiments the virus is West Nile. In some embodiments, the virus isDengue fever.

In some embodiments, the compounds of the invention are used to inhibitthe replication or infectivity of a virus. In some embodiments, thecompounds of the invention are used to inhibit the growth of a cellinfected with a virus. Examples of said viruses include, but are notlimited to, viruses of the orthmyxoviridae, paramyxoviridae,arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae,picornaviridae, and coronaviridae families. Specific examples of virusesinclude, but are not limited to, adenovirus, rhinovirus, hepatitis,immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, WestNile, small pox, encephalitis, yellow fever, Dengue fever, influenza(including human, avian, and swine), lassa, lymphocyticchoriomeningitis, junin, machuppo, guanarito, hantavirus, Rift ValleyFever, La Crosse, California encephalitis, Crimean-Congo, Marburg,Japanese Encephalitis, Kyasanur Forest, Venezuelan equine encephalitis,Eastern equine encephalitis, Western equine encephalitis, severe acuterespiratory syndrome (SARS), parainfluenza, respiratory syncytial, PuntaToro, Tacaribe and pachindae.

Thus, in some embodiments, the virus is selected from the groupconsisting of viruses of the orthmyxoviridae, paramyxoviridae,arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae,picornaviridae, and coronaviridae families. In some embodiments, theviral infection comprises a virus selected from the group consisting ofhepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie,Rhino, West Nile, small pox, encephalitis, yellow fever, Dengue fever,influenza (including human, avian, and swine), lassa, lymphocyticchoriomeningitis, junin, machuppo, guanarito, hantavirus, Rift ValleyFever, La Crosse, California encephalitis, Crimean-Congo, Marburg,Japanese Encephalitis, Kyasanur Forest, Venezuelan equine encephalitis,Eastern equine encephalitis, Western equine encephalitis, severe acuterespiratory syndrome (SARS), parainfluenza, respiratory syncytial, PuntaToro, Tacaribe and pachindae viruses.

In some embodiments, the viral infection comprises a virus selected fromthe group consisting of adenovirus, Dengue fever, influenza A andinfluenza B (including human, avian, and swine), junin, measles,parainfluenza, Pichinde, punta toro, respiratory syncytial, rhinovirus,Rift Valley Fever, severe acute respiratory syndrome (SARS), Tacaribe,Venezuelan equine encephalitis, West Nile and yellow fever viruses.

In some embodiments, the virus is Ebola, Marburg, yellow fever,influenza A or influenza B. In some embodiments, the virus is Ebola. Insome embodiments, the virus is Marburg. In some embodiments, the virusis yellow fever. In some embodiments, the virus is influenza A orinfluenza B.

In some embodiments, the virus is West Nile or Dengue fever. In someembodiments the virus is West Nile. In some embodiments, the virus isDengue fever.

In some embodiments, the present invention provides a method forinhibiting a viral RNA or DNA polymerase in a subject comprisingadministering to said subject a therapeutically effective amount of acompound of formula I, or a pharmaceutically acceptable salt or hydratethereof.

According to the Baltimore classification system, RNA polymerase virusesmay be classified into groups such as, for example, double-strandedviruses, positive-sense single-stranded viruses, and negative-sensesingle stranded viruses. Positive-sense single-stranded familiesinclude, for example, coronaviridae, picornaviridae, togaviridae,flaviviridae, and the like. Negative-sense single-stranded familiesinclude, for example, paramyxoviridae, arenaviridae, bunyaviridae,orthomyxoviridae, filoviridae, and the like. Each of the virus familiesmay be further classified into genera, species, and serotype (orsubtype). Other designations for taxonomic designations of viruses areset forth by the classification guidelines according to theInternational Committee on Taxonomy of Viruses.

In some embodiments, the RNA polymerase is double-stranded. In someembodiments, the RNA polymerase is single-stranded. In some embodiments,the RNA polymerase is positive-sense single-stranded. In someembodiments, the RNA polymerase is negative-sense single-stranded.

In some embodiments, the methods of the present invention provide forbroad spectrum inhibition of viruses and/or RNA polymerases from one ormore family, genus, subtype, strain and/or serotype of virus. In someembodiments, the methods provide for broad spectrum treatment,suppression, or prevention of infection from one or more family, genus,subtype, strain, or serotype of virus. In some embodiments, the broadspectrum encompasses more than two families, genera, subtypes, strainsand/or serotypes of virus.

In some embodiments, the present invention provides a method forinhibiting viral polymerases from one or more virus family, genus,subtype, serotype, or strain. In some embodiments, the present inventionprovides a method for treating, suppressing, and/or preventing a viralinfection wherein the viral infection is a result of infection with oneor more virus family, genus, subtype, serotype, or strain.

In some embodiments, the viral polymerases or viruses are from one ormore virus genus. In some embodiments, the viral polymerases or virusesare from one or more virus species. In some embodiments, the viralpolymerases or viruses are selected from one or more subtypes orserotypes. In some embodiments, the viral polymerases or viruses areselected from one or more strains.

In some embodiments, the RNA viral polymerase is selected from the groupconsisting of polymerases of the orthmyxoviridae, paramyxoviridae,arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae,picornaviridae, and coronaviridae families. In some embodiments, the RNAviral polymerase is selected from the group consisting of polymerases ofthe orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae,flaviviridae, and coronaviridae families. In some embodiments, the RNAviral polymerase comprises a polymerase selected from the groupconsisting of hepatitis, immunodeficiency virus, polio, measles, Ebola,Coxsackie, Rhino, West Nile, small pox, encephalitis, yellow fever,Dengue fever, influenza (including human, avian, and swine), lassa,lymphocytic choriomeningitis, junin, machuppo, guanarito, hantavirus,Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo,Marburg, Japanese Encephalitis, Kyasanur Forest, Venezuelan equineencephalitis, Eastern equine encephalitis, Western equine encephalitis,severe acute respiratory syndrome (SARS), parainfluenza, respiratorysyncytial, Punta Toro, Tacaribe and pachindae viral polymerase.

In some embodiments, the RNA viral polymerase is selected from the groupconsisting of adenovirus, Dengue fever, influenza A and influenza B(including human, avian, and swine), junin, measles, parainfluenza,Pichinde, punta toro, respiratory syncytial, rhinovirus, Rift ValleyFever, severe acute respiratory syndrome (SARS), Tacaribe, Venezuelanequine encephalitis, West Nile and yellow fever viral polymerase.

In some embodiments, the RNA viral polymerase is Ebola, Marburg, yellowfever, influenza A or influenza B viral polymerase. In some embodiments,the RNA viral polymerase is Ebola viral polymerase. In some embodiments,the RNA viral polymerase is Marburg viral polymerase. In someembodiments, the RNA viral polymerase is yellow fever viral polymerase.In some embodiments, the RNA viral polymerase is influenza A viralpolymerase or influenza B viral polymerase. In some embodiments, theviral polymerase is West Nile or Dengue fever viral polymerase. In someembodiments the viral polymerase is West Nile viral polymerase. In someembodiments, the viral polymerase is Dengue fever viral polymerase.

In some embodiments, the viruses are selected from the group consistingof orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae,flaviviridae, filoviridae, togaviridae, picornaviridae, andcoronaviridae families. In some embodiments, the viruses are selectedfrom the group consisting of hepatitis, immunodeficiency virus, polio,measles, Ebola, Coxsackie, Rhino, West Nile, small pox, encephalitis,yellow fever, Dengue fever, influenza (including human, avian, andswine), lassa, lymphocytic choriomeningitis, junin, machuppo, guanarito,hantavirus, Rift Valley Fever, La Crosse, California encephalitis,Crimean-Congo, Marburg, Japanese Encephalitis, Kyasanur Forest,Venezuelan equine encephalitis, Eastern equine encephalitis, Westernequine encephalitis, severe acute respiratory syndrome (SARS),parainfluenza, respiratory syncytial, Punta Toro, Tacaribe andpachindae.

In some embodiments, the viruses are selected from the group consistingof adenovirus, Dengue fever, influenza A and influenza B (includinghuman, avian, and swine), junin, measles, parainfluenza, Pichinde, puntatoro, respiratory syncytial, rhinovirus, Rift Valley Fever, severe acuterespiratory syndrome (SARS), Tacaribe, Venezuelan equine encephalitis,West Nile and yellow fever viruses.

In some embodiments, the virus is Ebola, Marburg, yellow fever,influenza A or influenza B. In some embodiments, the virus is Ebola. Insome embodiments, the virus is Marburg. In some embodiments, the virusis yellow fever. In some embodiments, the virus is influenza A orinfluenza B.

In some embodiments, the virus is West Nile or Dengue fever. In someembodiments the virus is West Nile. In some embodiments, the virus isDengue fever.

The genome of the influenza A virus has an RNA-dependent RNA polymerase,which catalyzes viral RNA transcription and replication. Because thetranscription and replication of the virus depends on the activity ofRNA polymerase, this enzyme has become of interest as a target fordevelopment of new anti-viral compounds in the wake of the recentemergence of drug resistant viruses. Viruses may develop resistance toone drug upon treatment, thus decreasing the efficacy of the drug andrequiring the subject to be treated with another antiviral drug. A drugor treatment that exhibits simultaneous efficacy against a broadspectrum of viral strains would thus be useful.

In addition, the composition or method may further comprise one or moreadditional anti-viral agents in combination with a compound of formulaI. Examples of such anti-viral agents include, but are not limited to,Cytovene, Ganciclovir, trisodium phosphonoformate, ribavirin,interferon, d4T, ddI, AZT, and Amantadine, Rimandatine, and otheranti-influenza agents; Acyclovir, and related agents, Foscarnet andother anti-herpes virus agents. Non-limiting examples of neuraminidaseinhibitors include laninamivir, oseltamivir, zanamivir, and peramivir.

Compounds that relate to inhibition of influenza polymerase aredescribed, for example, in U.S. Pat. Nos. 7,388,002; 7,560,434; and inU.S. patent application Ser. Nos. 12/440,697 (published as U.S. PatentPublication No. 20100129317); and Ser. No. 12/398,866 (published as U.S.Patent Publication No. 20090227524), each of which herein incorporatedby reference in its entirety. Currently, there is one influenzapolymerase inhibitor in clinical trials, known as T-705 (favipiravir;6-fluoro-3-hydroxy-2-pyrazinecarboxamide). T-705 possesses potent andbroad spectrum antiviral activity against multiple strains of influenzavirus infection in vitro and in vivo (Kiso et al., PNAS 2010, 107,882-887; herein incorporated by reference in its entirety). T-705 ischaracterized by a mechanism of action that is different from mostanti-influenza viral drugs.

Another class of compounds used as anti-virals are M2 inhibitors (See,Pielak, R., Schnell, J., & Chou, J. (2009) Proceedings of the NationalAcademy of Sciences, 106 (18), 7379-7384 (herein incorporated byreference in its entirety). Exemplary members of this class includeamantadine and rimantadine.

Thus, in some embodiments, the methods of the invention further compriseadministration of one or more additional anti-viral agents.

In some embodiments, an additional anti-viral agent is selected from thegroup consisting of Cytovene, Ganciclovir, trisodium phosphonoformate,ribavirin, interferon, d4T, ddI, AZT, and amantadine, rimandatine, T-705and other anti-influenza agents; Acyclovir, and related agents,Foscarnet and other anti-herpes virus agents.

In some embodiments, an additional anti-viral agent is an anti-influenzaagent. In some embodiments, an additional anti-viral agent is aneuraminidase inhibitor. In some embodiments, an additional anti-viralagent is selected from the group consisting of laninamivir, oseltamivir,zanamivir, and peramivir. In some embodiments, an additional anti-viralagent is paramivir. In some embodiments, an additional anti-viral agentis laninamivir. In some embodiments, an additional anti-viral agent isoseltamivir. In some embodiments, an additional anti-viral agent iszanamivir.

In some embodiments, an additional anti-viral agent is an M2 inhibitor.In some embodiments, an additional anti-viral agent is selected from thegroup consisting of amantadine and rimandatine.

In some embodiments, the methods of the invention compriseadministration of two additional anti-viral agents. In some embodiments,the additional anti-viral agents are a neuraminidase inhibitor and an M2inhibitor. In some embodiments, the additional anti-viral agents areselected from the groups consisting of 1) laninamivir, oseltamivir,zanamivir, and peramivir; and 2) amantadine and rimandatine. In someembodiments, the additional antiviral agents are peramivir andamantadine. In some embodiments, the additional antiviral agents areperamivir and rimantadine.

The present invention provides methods for inhibiting a viral RNA or DNApolymerase comprising contacting the polymerase with an effectiveinhibitory amount of the compound of formula I, or a pharmaceuticallyacceptable salt or solvate thereof.

In some embodiments, the present invention provides a method fortreating a subject suffering from a viral infection comprisingadministering to said subject a therapeutically effective amount of acompound of formula I, or pharmaceutically acceptable salt or hydratethereof.

In some embodiments, the present invention provides a method forsuppressing a viral infection in a subject comprising administering tothe subject a therapeutically effective amount of a compound of formulaI, or pharmaceutically acceptable salt or hydrate thereof.

In some embodiments, the present invention provides a method fortreating a subject suffering from an RNA viral infection which comprisesadministering to said subject a therapeutically effective amount of acompound of formula I, or pharmaceutically acceptable salt or solvatethereof.

In some embodiments, the viral infection is comprises infection by oneor more viruses.

In some embodiments, the viral infections are infections selected fromviruses of the orthmyxoviridae, paramyxoviridae, arenaviridae,bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, orcoronaviridae families, or any combination thereof. In some embodiments,the viral infections are infections selected from viruses of hepatitis,immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, WestNile, small pox, encephalitis, yellow fever, Dengue fever, influenza(including human, avian, and swine), lassa, lymphocyticchoriomeningitis, junin, machuppo, guanarito, hantavirus, Rift ValleyFever, La Crosse, California encephalitis, Crimean-Congo, Marburg,Japanese Encephalitis, Kyasanur Forest, Venezuelan equine encephalitis,Eastern equine encephalitis, Western equine encephalitis, severe acuterespiratory syndrome (SARS), parainfluenza, respiratory syncytial, PuntaToro, Tacaribe and pachindae, or any combination thereof

In some embodiments, the viral infection comprises infection by one ormore viruses selected from the group consisting of adenovirus, Denguefever, influenza A and influenza B (including human, avian, and swine),junin, measles, parainfluenza, Pichinde, punta toro, respiratorysyncytial, rhinovirus, Rift Valley Fever, severe acute respiratorysyndrome (SARS), Tacaribe, Venezuelan equine encephalitis, West Nile andyellow fever viruses.

In some embodiments, the virus is Ebola, Marburg, yellow fever,influenza A or influenza B. In some embodiments, the virus is Ebola. Insome embodiments, the virus is Marburg. In some embodiments, the virusis yellow fever. In some embodiments, the virus is influenza A orinfluenza B.

In some embodiments, the virus is West Nile or Dengue fever. In someembodiments the virus is West Nile. In some embodiments, the virus isDengue fever.

In some embodiments, the viral infections are infections selected fromviruses of influenza A, influenza B, PIV, RSV, Junin, Pichinde, RiftValley Fever, Dengue Fever, measles, Yellow Fever, and SARS-CoV, or anycombination thereof. In some embodiments, the viral infections areinfections selected from influenza A and B, subtypes thereof, strainsthereof, or any combination thereof. In some embodiments, the viralinfections are infections selected from Ebola, Marburg, or yellow fever.In some embodiments, the viral infection is Ebola. In some embodiments,the viral infection is Marburg. In some embodiments, the viral infectionis yellow fever. In some embodiments, the viral infection is West Nileor Dengue fever. In some embodiments the viral infection is West Nile.In some embodiments, the viral infection is Dengue fever.

In some embodiments, the disclosure provides for the use ofpharmaceutical compositions and/or medicaments comprised of the compoundof formula I, or a pharmaceutically acceptable salt or hydrate thereof,in a method of treating a viral infection, and/or disease state, and/orcondition caused by or related to such viral infection.

In some embodiments, the method of treatment comprises the steps of: i)identifying a subject in need of such treatment; (ii) providing acompound of formula I, or a pharmaceutically acceptable salt or hydratethereof, or a composition comprising a compound of formula I, or apharmaceutically acceptable salt or hydrate thereof; and (iii)administering said compound or composition in a therapeuticallyeffective amount to treat the viral infection in the subject or toinhibit the activity of viral DNA or RNA polymerase in a subject in needof such treatment.

In some embodiments, the treatment efficacy results from the inhibitionof a viral DNA or RNA polymerase. In some embodiments, the treatmentefficacy results from inhibiting viral polymerases from one or morevirus family.

In some embodiments, the viral polymerases or viruses are from one ormore virus genus. In some embodiments, the viral polymerases or virusesare from one or more virus species. In some embodiments, the viralpolymerases or viruses are selected from one or more subtype, serotype,or strain.

In some embodiments, the method is performed in vivo.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is avian. In someembodiments, the subject is a swine or pig.

In some embodiments, the bodily fluid is blood. In some embodiments, thebodily fluid is plasma. In some embodiments, the bodily fluid is bloodserum.

In some embodiments, the compound or composition is administeredintravenously, interperitonealy, intramuscularly or orally.

In some embodiments, the compound or composition is administeredintravenously.

In some embodiments, the compound or composition is administeredintraperitonealy.

In some embodiments, the compound or composition is administeredintramuscularly.

In some embodiments, the compound or composition is administered orally.

The methods comprise administering to the subject a therapeuticallyeffective amount of a compound of formula I, or a pharmaceuticallyacceptable salt thereof, or a composition comprising a compound offormula I, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. The pharmaceutically acceptablecarriers are well-known to those skilled in the art, and include, forexample, adjuvants, diluents, excipients, fillers, lubricants andvehicles. Often, the pharmaceutically acceptable carrier is chemicallyinert toward the active compounds and is non-toxic under the conditionsof use. Examples of pharmaceutically acceptable carriers may include,for example, water or saline solution, polymers such as polyethyleneglycol, carbohydrates and derivatives thereof, oils, fatty acids, oralcohols. In some embodiments, the carrier is saline or water. In someembodiments, the carrier is saline. In some embodiments, the carrier iswater.

In some embodiments, the method of prevention or suppression of theviral infection or disease state comprises the steps of: i) identifyinga subject in need of such treatment; (ii) providing a compound offormula I, or a pharmaceutically acceptable salt or hydrate thereof, ora composition comprising a compound of formula I, or a pharmaceuticallyacceptable salt or hydrate thereof; and (iii) administering saidcompound or composition in a therapeutically effective amount to preventor suppress the viral infection or disease state in the subject or toinhibit the activity of viral DNA or RNA polymerase in a subject in needof such treatment.

The compounds of the present invention are prepared in different forms,such as salts, hydrates, solvates, tautomers or complexes, and theinvention includes methods encompassing all variant forms of thecompounds.

In some embodiments, the methods of the invention comprisepharmaceutically acceptable salts of the compound of formula I. Acompound of formula I also may be formulated as a pharmaceuticallyacceptable salt, e.g., acid addition salt, and complexes thereof. Thepreparation of such salts can facilitate the pharmacological use byaltering the physical characteristics of the agent without preventingits physiological effect. Examples of useful alterations in physicalproperties include, but are not limited to, lowering the melting pointto facilitate transmucosal administration and increasing the solubilityto facilitate administering higher concentrations of the drug.

The subjects of the invention are in vitro and in vivo systems,including, for example, isolated or cultured cells or tissues,non-cellular in vitro assay systems and animals (e.g., an amphibian, abird, a fish, a mammal, a marsupial, a human, a domestic animal such as,for example, a cat, dog, monkey, mouse or rat; or a commercial animalsuch as, for example, a cow or pig).

The compounds of the invention may be formulated into pharmaceuticalcompositions for administration to subjects in a biologically compatibleform suitable for administration in vivo. According to another aspect,the present invention provides a pharmaceutical composition comprisingcompounds of formula I in admixture with a pharmaceutically acceptablediluent and/or carrier. The pharmaceutically-acceptable carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof. Thepharmaceutically-acceptable carriers employed herein may be selectedfrom various organic or inorganic materials that are used as materialsfor pharmaceutical formulations and which are incorporated as analgesicagents, buffers, binders, disintegrants, diluents, emulsifiers,excipients, extenders, glidants, solubilizers, stabilizers, suspendingagents, tonicity agents, vehicles and viscosity-increasing agents.Pharmaceutical additives, such as antioxidants, aromatics, colorants,flavor-improving agents, preservatives, and sweeteners, may also beadded. Examples of acceptable pharmaceutical carriers includecarboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic,lactose, magnesium stearate, methyl cellulose, powders, saline, sodiumalginate, sucrose, starch, talc and water, among others. In someembodiments, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans.

Surfactants such as, for example, detergents, are also suitable for usein the formulations. Specific examples of surfactants includepolyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetateand of vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol,glycerol, sorbitol or polyoxyethylenated esters of sorbitan; lecithin orsodium carboxymethylcellulose; or acrylic derivatives, such asmethacrylates and others, anionic surfactants, such as alkalinestearates, in particular sodium, potassium or ammonium stearate; calciumstearate or triethanolamine stearate; alkyl sulfates, in particularsodium lauryl sufate and sodium cetyl sulfate; sodiumdodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fattyacids, in particular those derived from coconut oil, cationicsurfactants, such as water-soluble quaternary ammonium salts of formulaN⁺R′R″R′″R″″Y⁻, in which the R radicals are identical or differentoptionally hydroxylated hydrocarbon radicals and Y⁻ is an anion of astrong acid, such as halide, sulfate and sulfonate anions;cetyltrimethylammonium bromide is one of the cationic surfactants whichcan be used, amine salts of formula N⁺R′R″R′″, in which the R radicalsare identical or different optionally hydroxylated hydrocarbon radicals;octadecylamine hydrochloride is one of the cationic surfactants whichcan be used, non-ionic surfactants, such as optionallypolyoxyethylenated esters of sorbitan, in particular Polysorbate 80, orpolyoxyethylenated alkyl ethers; polyethylene glycol stearate,polyoxyethylenated derivatives of castor oil, polyglycerol esters,polyoxyethylenated fatty alcohols, polyoxyethylenated fatty acids orcopolymers of ethylene oxide and of propylene oxide, amphotericsurfactants, such as substituted lauryl compounds of betaine,

When administered to a subject, the compounds of formula I andpharmaceutically acceptable carriers may be sterile. In someembodiments, water is a carrier when the compound of formula I isadministered intravenously. In some embodiments, the carrier is a salinesolution when the compound of formula I is administered intravenously.Aqueous dextrose and glycerol solutions may also be employed as liquidcarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers may also include excipients such as starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, polyethylene glycol 300, water, ethanol, polysorbate20, and the like. The present compositions, if desired, may also containminor amounts of wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical formulations of the present invention are prepared bymethods well-known in the pharmaceutical arts. For example, thecompounds of formula I are brought into association with a carrierand/or diluent, as a suspension or solution. Optionally, one or moreaccessory ingredients (e.g., buffers, flavoring agents, surface activeagents, and the like) also are added. The choice of carrier isdetermined by the solubility and chemical nature of the compounds,chosen route of administration and standard pharmaceutical practice. Insome embodiments, the formulation comprises a compound of formula I andwater. In some embodiments, the formulation comprises a compound offormula I and saline.

Additionally, the compounds of the present invention are administered toa human or animal subject by known procedures including, withoutlimitation, oral administration, sublingual or buccal administration,parenteral administration, transdermal administration, via inhalation orintranasally, vaginally, rectally, and intramuscularly. The compounds ofthe invention are administered parenterally, by epifascial,intracapsular, intracranial, intracutaneous, intrathecal, intramuscular,intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular,intravenous, parenchymatous, subcutaneous or sublingual injection, or byway of catheter. In some embodiments, the compound is administered tothe subject by way of intramuscular delivery. In some embodiments, thecompound is administered to the subject by way of intraperitonealdelivery. In some embodiments, the compound is administered to thesubject by way of intravenous delivery. In some embodiments, thecompound is administered orally.

For oral administration, a formulation of the compounds of the inventionmay be presented as capsules, tablets, powders, granules, or as asuspension or solution. Capsule formulations may be gelatin, soft-gel orsolid. Tablets and capsule formulations may further contain one or moreadjuvants, binders, diluents, disintegrants, excipients, fillers, orlubricants, each of which are known in the art. Examples of such includecarbohydrates such as lactose or sucrose, dibasic calcium phosphateanhydrous, corn starch, mannitol, xylitol, cellulose or derivativesthereof, microcrystalline cellulose, gelatin, stearates, silicondioxide, talc, sodium starch glycolate, acacia, flavoring agents,preservatives, buffering agents, disintegrants, and colorants. Orallyadministered compositions may contain one or more optional agents suchas, for example, sweetening agents such as fructose, aspartame orsaccharin; flavoring agents such as peppermint, oil of wintergreen, orcherry; coloring agents; and preservative agents, to provide apharmaceutically palatable preparation.

For parenteral administration (i.e., administration by injection througha route other than the alimentary canal), the compounds of the inventionmay be combined with a sterile aqueous solution that is isotonic withthe blood of the subject. Such a formulation is prepared by dissolving asolid active ingredient in water containing physiologically-compatiblesubstances, such as sodium chloride, glycine and the like, and having abuffered pH compatible with physiological conditions, so as to producean aqueous solution, then rendering said solution sterile. Theformulation may be presented in unit or multi-dose containers, such assealed ampules or vials. The formulation may be delivered by any mode ofinjection, including, without limitation, epifascial, intracapsular,intracranial, intracutaneous, intrathecal, intramuscular, intraorbital,intraperitoneal, intraspinal, intrasternal, intravascular, intravenous,parenchymatous, subcutaneous, or sublingual or by way of catheter intothe subject's body.

Parenteral administration includes aqueous and non-aqueous basedsolutions. Examples of which include, for example, water, saline,aqueous sugar or sugar alcohol solutions, alcoholic (such as ethylalcohol, isopropanol, glycols), ethers, oils, glycerides, fatty acids,and fatty acid esters. In some embodiments, water is used for parenteraladministration. In some embodiments, saline is used for parenteraladministration. Oils for parenteral injection include animal, vegetable,synthetic or petroleum based oils. Examples of sugars for solutioninclude sucrose, lactose, dextrose, mannose, and the like. Examples ofoils include mineral oil, petrolatum, soybean, corn, cottonseed, peanut,and the like. Examples of fatty acids and esters include oleic acid,myristic acid, stearic acid, isostearic acid, and esters thereof.

For transdermal administration, the compounds of the invention arecombined with skin penetration enhancers, such as propylene glycol,polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone and the like, which increase the permeability of theskin to the compounds of the invention and permit the compounds topenetrate through the skin and into the bloodstream. Thecompound/enhancer compositions also may be further combined with apolymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which are dissolved in a solvent, such asmethylene chloride, evaporated to the desired viscosity and then appliedto backing material to provide a patch.

In some embodiments, the composition is in unit dose form such as atablet, capsule or single-dose vial. Suitable unit doses, i.e.,therapeutically effective amounts, may be determined during clinicaltrials designed appropriately for each of the conditions for whichadministration of a chosen compound is indicated and will, of course,vary depending on the desired clinical endpoint.

The present invention also provides articles of manufacture for treatingand preventing disorders, such as viral disorders, in a subject. Thearticles of manufacture comprise a pharmaceutical composition of thecompounds of formula I, optionally further containing at least oneadditional antiviral compound, as described herein. The articles ofmanufacture are packaged with indications for various disorders that thepharmaceutical compositions are capable of treating and/or preventing.For example, the articles of manufacture comprise a unit dose of acompound disclosed herein that is capable of treating or preventing acertain disorder, and an indication that the unit dose is capable oftreating or preventing a certain disorder, for example a viralinfection.

In accordance with a method of the present invention, the compounds offormula I are administered to the subject (or are contacted with cellsof the subject) in an amount effective to limit or prevent a decrease inthe level of virus in the subject, particularly in cells of the subject.This amount is readily determined by the skilled artisan, based uponknown procedures, including analysis of titration curves established invivo and methods and assays disclosed herein. In some embodiments, asuitable amount of the compounds of the invention effective to limit orprevent an increase in the level of viral particles in the subjectranges from about 0.01 mg/kg/day to about 1000 mg/kg/day, and/or is anamount sufficient to achieve plasma levels ranging from about 300 ng/mLto about 1000 ng/mL or greater. In some embodiments, the amount ofcompounds from the invention ranges from about 5 mg/kg/day to about 1000mg/kg/day. In some embodiments, from about 0.01 mg/kg/day to about 500mg/kg/day is administered. In some embodiments, from about 0.01mg/kg/day to about 300 mg/kg/day is administered. In some embodiments,from about 0.01 mg/kg/day to about 200 mg/kg/day is administered. Insome embodiments, from about 0.05 mg/kg/day to about 100 mg/kg/day isadministered. In some embodiments, from about 0.05 mg/kg/day to about 50mg/kg/day is administered. In some embodiments, from about 0.05mg/kg/day to about 30 mg/kg/day is administered. In some embodiments,from about 0.05 mg/kg/day to about 10 mg/kg/day is administered.

The precise dose to be employed in the compositions will also depend onthe route of administration, and the seriousness of the infection ordisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. However, suitableeffective dosage ranges for intramuscular administration are generallyabout 0.5 to about 1000 mg of the compound of formula I per kilogrambody weight. In specific embodiments, the i.m. dose is about 500 toabout 1000 mg/kg, about 300 to about 500 mg/kg, about 200 to about 300mg/kg, about 100 to about 200 mg/kg, about 50 to about 100 mg/kg, about10 to about 50 mg/kg, or about 5 to about 10 mg/kg (or the equivalentdoses expressed per square meter of body surface area). Alternatively, asuitable dose range for i.v. administration may be obtained using dosesof about 5 to about 1000 mg, without adjustment for a patient's bodyweight or body surface area. Alternatively, a suitable dose range fori.p. administration may be obtained using doses of about 5 to about 1000mg, without adjustment for a patient's body weight or body surface area.Oral compositions may contain about 10% to about 95% by weight of one ormore compound of formula I alone or in combination with anothertherapeutic agent. In some embodiments of the invention, suitable doseranges for oral, i.p., or i.m. administration are generally about 5 toabout 1000 mg, preferably about 5 to about 500 mg of compound perkilogram body weight or their equivalent doses expressed per squaremeter of body surface area. In some embodiments the oral, i.p., or i.m.dose is about 5 to about 50 mg/kg, about 50 to about 80 mg/kg, about 80to about 150 mg/kg, about 150 to about 250 mg/kg, about 250 to about 350mg/kg, about 350 to about 450 mg/kg, about 450 to about 550 mg/kg, about550 to about 700 mg/kg, about 700 to about 1000 mg/kg (or the equivalentdoses expressed per square meter of body surface area). In someembodiments, a suitable dose range for oral, i.p., or i.m.administration is from about 5 to about 2000 mg, without adjustment fora patient's body weight or body surface area. Other effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems. Such animal models and systems are well knownin the art.

In certain aspects, an “effective amount” of a compound in the contextof a viral infection is an amount sufficient to reduce one or more ofthe following steps of a the life cycle of a virus: the docking of thevirus particle to a cell, the introduction of viral genetic informationinto a cell, the expression of viral proteins, the production of newvirus particles and the release of virus particles from a cell by atleast 5%, preferably at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. Insome embodiments, an effective amount of a compound in the context of aviral infection reduces the replication, multiplication or spread of avirus by at least 5%, preferably at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or 100%. In some embodiments, an effective amount of a compound inthe context of a viral infection increases the survival rate of infectedsubjects by at least 5%, preferably at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or 100%.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be within the scope of the present invention.

The invention is further described by the following non-limitingExamples.

EXAMPLES Example 1 Synthesis of(2S,3S,4R,5R)-2-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol [Compound 1 (Formula I, Wherein A=NH₂ and B═H) asthe HCl Salt]

Step-1:

To a solution of7-((2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-2-yl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one(1-1) [(prepared according to procedure reported by Evans, Gary B.;Furneaux, Richard H.; Hutchison, Tracy L.; Kezar, Hollis S.; Morris,Philip E., Jr.; Schramm, Vern L.; Tyler, Peter C in Journal of OrganicChemistry (2001), 66(17), 5723-5730; herein incorporated by reference inits entirety) 115 g, 390 mmol] in water and methanol (1:1, 2.4 L) wasadded triethylamine (113 mL, 1.12 mol) at room temperature followed by(Boc)₂O (227 g, 1.04 mol). The reaction mixture was stirred at roomtemperature overnight. The solid product was collected by filtration,washed with water, and dried in vacuum to afford(2R,3R,4S,5S)-tert-butyl3,4-dihydroxy-2-(hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate(1-2) (100%) as a white solid. ¹H NMR (300 MHz, DMSO) δ 7.85 (s, 1H),7.35 (s, 1H), 4.73-4.53 (m, 1H), 4.29 (s, 1H), 4.03 (s, 1H), 3.97 (s,1H), 3.70-3.53 (m, 2H), 1.36 and 1.04 (s, 3H, 6H for rotomers).

Step-2:

To a solution of (2R,3R,4S,5S)-tert-butyl3,4-dihydroxy-2-(hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate(1-2) in pyridine (184 mmol, 2.26 mol) was added DMAP (0.79 g, 6.46mmol) and acetic anhydride (107 mL, 1131 mmol) at room temperature. Thereaction mixture was stirred at room temperature overnight. The reactionmixture was diluted with chloroform and washed with water, aqueous HCl,water, and aqueous saturated sodium bicarbonate. The organic layer wasdried, filtered and concentrated in vacuum, to furnish(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-3) (150 g), which was pure enough to be used as such fornext step. MS (ES⁺) 493.1 (M+1), 515.1 (M+Na); (ES⁻) 491.4 (M−1).

Step-3:

To a solution of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-3) (150 g, 300 mmol) in acetonitrile (660 mL) was addedbenzyltriethylammonium chloride (137 g, 600 mmol), dimethylaniline (57mL, 450 mmol), followed by POCl₃ (164 mL, 1800 mmol) at roomtemperature. The reaction mixture was heated at 80° C. for 1 h. Thereaction mixture was cooled to room temperature and concentrated todryness under vacuum. The residue obtained was dissolved in chloroformand washed with aqueous saturated sodium bicarbonate, brine, dried,filtered and concentrated to dryness. The residue of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-4) was used as such in next step without purification. ¹HNMR (300 MHz, DMSO) δ 12.54 (s, 1H), 8.65 (s, 1H), 7.92 (s, 1H), 5.85(m, 1H), 5.45 (m, 1H), 5.10 (m, 1H), 4.49 (m, 2H), 4.07 (m, 1H),2.07-1.99 (m, 9H), 1.19 (2 bs, 9H, rotomers).

Step-4:

To a solution of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-4) (300 mmol) in DMF (540 mL) was added sodium azide (97.5g, 1500 mmol) and heated at 80° C. overnight. The reaction mixture wasconcentrated in vacuum and the residue obtained was dissolved inchloroform. The chloroform layer was washed with water, dried, filteredand concentrated in vacuum. Purification by crystallization from(acetone:hexane=1:2) furnished(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-5). ¹H NMR (300 MHz, DMSO) δ 13.56-13.00 (bs, 1H), 9.86 (s,1H), 7.95 (s, 1H), 5.78 (m, 1H), 5.40 (m, 1H), 5.26-5.14 (m, 1H), 4.54(m, 1H), 4.42 (m, 1H), 4.16-4.03 (m, 1H), 2.06 (s, 3H), 2.02 (s, 6H),1.14 (bs, 9H); MS (ES⁺) 540.0 (M+1); (ES⁻) 515.9 (M−1).

Step-5:

To a solution of(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-5) (300 mmol) in methanol (1 L) was added Pd(OH)₂ (30 g).The reaction mixture was hydrogenated at (160 psi) overnight, andfiltered to remove catalyst through celite. The filtrate wasconcentrated in vacuum to furnish(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-6) (113 g). ¹H NMR (300 MHz, DMSO) δ 12.47-11.92 (m, 1H),8.84-8.03 (m, 3H), 7.90-7.68 (m, 1H), 5.70-5.51 (m, 1H), 5.38 (m, 1H),5.12 (m, 1H), 4.42 (m, 2H), 4.17-4.00 (m, 1H), 2.07 (s, 3H), 2.05 (s,3H), 2.00 (s, 3H), 1.14 (s, 9H); MS (ES⁺) 492.1 (M+1), (ES⁻) 490.0(M−1).

Step-6:

To a solution of(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-6) (111 g, 226 mmol) in methanol (500 mL) was added NaOMe(25% w/w in methanol, 4.88 g, 22.6 mmol) at room temperature. Thereaction mixture was stirred at room temperature for 3 h andconcentrated in vacuum to give (2S,3S,4R,5R)-tert-butyl2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate(1-7). ¹H NMR (300 MHz, DMSO) δ 11.40-10.73 (bs, 1H), 8.01 (s, 1H), 7.39(2s, 1H), 6.90 (s, 2H), 4.83 (m, 2H), 4.45 (m, 2H), 3.96 (s, 2H), 3.58(m, 3H), 1.31 and 0.99(s, 3H, 6H, rotomers); MS (ES⁺) 366.0 (M+1), 388.0(M+Na); (ES⁻) 363.8 (M−1).

Step-7:

A solution of (2S,3S,4R,5R)-tert-butyl2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate(1-7) aqueous HCl (160 mL of conc. HCl and 400 mL of water) was stirredat room temperature for 30 min and then concentrated in vacuum todryness. The residue obtained was dissolved in water, treated withactivated charcoal and refluxed for 30 min. The hot solution wasfiltered through celite and concentrated in vacuum to obtain asemi-solid product, which was recrystallized from water and ethanol tofurnish(2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol(1) (50 g, overall yield for 7 steps: 42.6%) as white crystal. ¹H NMR(300 MHz, D₂O) δ 8.41 (s, 1H), 8.02 (s, 1H), 4.99 (d, J=9 Hz, 1H), 4.78(m, 1H), 4.45 (dd, J=3, 1.5 Hz, 1H), 3.97 (m, 2H), 3.90 (m, 1H); MS(ES⁺) 266.2 (M+1), (ES⁻) 264.0 (M−1); Analysis: Calculated forC₁₁H₁₅N₅O₃.2 HCl: C, 39.07; H, 5.07; N, 20.71; Cl, 20.97; Found: C,39.09; H, 5.10; N, 20.49; Cl, 20.84.

Example 2 Large-Scale Synthesis of(2S,3S,4R,5R)-2-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol [Compound 1 (Formula I, Wherein A=NH₂ and B=H) asthe HCl Salt]

Step-1:

To a suspension of7-((2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-2-yl)-3H-pyrrolo[3,2-d]pyrimidin-4(5H)-one(1-1) [(prepared according to procedure reported by Evans, Gary B.;Furneaux, Richard H.; Hutchison, Tracy L.; Kezar, Hollis S.; Morris,Philip E., Jr.; Schramm, Vern L.; Tyler, Peter C in Journal of OrganicChemistry (2001), 66(17), 5723-5730), 500.0 g, 1.474 mol, 1 eq)] in awater : methanol mixture (1:1, 10.4 L) was added triethylamine (621 mL,4.422 mol, 3.0 eq) at room temperature followed by (Boc)₂O (987 g, 4.53mol, 3.1 eq). The reaction mixture became a clear colored solution afterthe addition of (Boc)₂O with slight increase of the internal temperaturefrom 28° C. to 33° C. The solution started showing some turbidity after1 hour of stirring. The solution was stirred at room temperatureovernight. The solid product was collected by filtration and washed withwater (5.0 L), dried at high vacuum at 50° C. to furnish(2R,3R,4S,5S)-tert-butyl3,4-dihydroxy-2-(hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate(1-2) (482 g, 89%).

¹H NMR (300 MHz, DMSO) δ 11.92 (s, 2H), 7.81 (s, 1H), 7.32 (d, J=22.7Hz, 1H), 5.73-5.20 (m, 1H), 5.05-4.91 (m, 1H), 4.87-4.76 (m, 1H),4.74-4.49 (m, 1H), 4.33-4.17 (m, 1H), 4.09-3.86 (m, 2H), 3.64-3.48 (m,2H), 1.39-1.00 (m, 9H); MS (ES+) 755.1 (2M+Na), (ES−) 731.7 (2M−1);Analysis; Calculated for C₁₆H₂₂N₄O₆: C, 52.45; H, 6.05; N, 15.29; Found:C, 52.24; H, 6.02; N, 15.05.

Step-2:

To a suspension of (2R,3R,4S,5S)-tert-butyl3,4-dihydroxy-2-(hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate(1-2) (482 g, 1.32 mole, 1.0 equiv.) in pyridine (740 mL, 9.21 mole, 7equiv.) was added DMAP (3.22 g, 26.32 mmol, 0.02 equiv.) and aceticanhydride (435 mL, 4.61 mmol, 3.5 eq) at room temperature. The internaltemperature started rising upon the addition of the acetic anhydridetherefore ice-water bath cooling was performed. Upon the total additionof the anhydride the temperature rose to 67° C. then decreased to roomtemperature. The ice water bath was removed after the reaction reached25° C. The suspension did not give a clear solution but a lightersuspension was observed. The reaction mixture was stirred at roomtemperature for 14 h to yield a non-clear solution. A worked-up aliquotshowed that there was no more starting material and two major spots byTLC (9:1 chloroform: methanol), MS shows two major peaks at (493.0, M+1)for product and tetraacetylated product (M+1=535). The reaction mixturewas diluted with 3.0 L of chloroform, stirred for 10 minutes then added2.0 L of deionized water. A waxy white product was formed in the aqueousorganic phase interface. This product remained in the aqueous phaseafter the partition was done. The organic phase was separated and washedagain with 2.0 L of water. The combined water washes were back extractedwith 1.0 of chloroform. The combined organic phases were washed withaqueous 2.0 N HCl (2×1.0 L), water (2×1.0 L), saturated sodiumbicarbonate (2×1.0 L) and brine (2×1.0 L). The organic layer was driedover MgSO₄, filtered and concentrated to dryness under vacuum and 50-55°C. water bath. The vacuum was switched to a high vacuum oil pump untilno more distillate was seen to furnish a dense syrupy product. Theproduct was left at high vacuum oil pump for 14 hrs to minimize theresidual pyridine. A combination of solid foam which turned into a nicewhite solid and a dense residue of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-3) was obtained (715, 110% yield). This percentage reflectsthe amount of tetraacetylated compound. The product was pure enough tobe used as is for next step. An analytical sample was prepared bypurification of the mixture using flash column chromatography [silicagel, eluting with 0-100% (9:1) ethyl acetate/methanol in hexane] tofurnish(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-3) as a white solid; ¹HNMR (300 MHz, DMSO) δ 12.13 (s, 1H,D2O Exchangeable), 11.98 (s, 1H, D₂O exchangeable), 7.82 (s, 1H), 7.29(s, 1H), 5.76 (s, 1H), 5.37 (t, J=4.5 Hz, 1H), 4.99 (s, 1H), 4.55 (dd,J=11.3, 6.6 Hz, 1H), 4.34 (d, J=8.3 Hz, 1H), 4.03 (q, J=7.1 Hz, 1H),2.01 (d, J=12.6 Hz, 9H), 1.23 (dd, J=39.9, 32.8 Hz, 9H); MS (ES+) 493.0(M+1); (ES−) 526.7 (M+Cl); Analysis: Calculated for C₂₂H₂₈N₄O₉: C,53.65; H, 5.73; N, 11.38; Found: C, 53.18; H, 5.89; N, 11.10

Step-3:

To a solution of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-3) (622 g, 1.26 mol, 1.0 eq) in acetonitrile (2.75 L) wasadded benzyltriethylammonium chloride (575 g, 2.5 mol, 2.0 eq),dimethylaniline (240 mL, 1.9 mol, 1.5 eq), followed by POCl₃ (706 mL,7.58 mol, 6.0 eq) at room temperature. A clear light yellow coloredsolution was obtained. The reaction mixture was slowly heated up to 80°C. and held at this temperature for 10 minutes. TLC in 9:1 chloroform:methanol shows that the reaction is >98% completed. The blackhomogeneous solution was cooled down to 50.0° C. and concentrated undervacuum (water bath 70-73° C.) to remove POCl₃ the residue was put underoil pump high vacuum until no more distillate was seen. The residue wasdissolved in 3.0 L of chloroform and quickly washed carefully withaqueous saturated sodium bicarbonate until a neutral pH was obtained.The organic layer was separated washed with water (2 L), brine (2 L),dried over MgSO₄, filtered and concentrated in vacuum to dryness (waterbath at 50-53° C.). The black product of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-4) was used as is in next step without purification.

Step-4:

To a solution of(2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyldiacetate (1-4) (622 g, 1.26 mol, 1 eq) in DMF (1.5 L) was added sodiumazide (411 g, 6.32 mol, 5 equiv.) and heated with stirring at 60° C. for10 hours at which time the reaction has gone to completion (TLC in 9:1chloroform methanol and 1:1 hexane: ethyl acetate). The reaction wascooled to 25° C., dumped in ice (2 L) and extracted with chloroform (2×1L). The chloroform layers were combined washed with water (2×2 L), brine(2 L), dried, filtered and concentrated in vacuum (water bath 70-80° C.)to yield a black sludge. Purification of the sludge was achieved bycolumn chromatography (987 g of black sludge, 8×30 inch column, ½ fullsilica gel, elution profile hexane:ethyl acetate; 9:1 (40.0L); 7:3(20.0L); 6:4 (20.0L); 1:1 (20L); 4:6 (20.0L) and 2:8 (20.0L); Theappropriate fractions were pooled and concentrated in vacuum (water bath50.0° C.) to furnish(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-5) (407.05 g, 62.3% yield for two steps) as a dense reddishcolored honey-like product. An analytical sample was prepared bypurification of the mixture by flash column chromatography [0-100% ethylacetate in hexane] to furnish(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-5) as an orange solid. ¹HNMR (300 MHz, DMSO) δ 13.08 (d,J=155.6 Hz, 1H, D₂O exchangeable), 9.86 (s, 1H), 7.61 (d, J=76.8 Hz,1H), 5.78 (t, J=4.5 Hz, 1H), 5.41 (t, J=4.3 Hz, 1H), 5.21 (s, 1H), 4.55(dd, J=11.4, 6.4 Hz, 1H), 4.41 (dd, J=11.4, 3.9 Hz, 1H), 4.07 (d, J=16.5Hz, 1H), 2.06 (s, 3H), 2.01 (d, J=9.9 Hz, 6H), 1.23 (dd, J=39.8, 32.7Hz, 9H); MS (ES+) 518.0 (M+1), 540 (M+23); (ES−) 516.4 (M−1); Analysis:Calculated for C₂₂H₂₇N₇O₈: C, 51.06; H, 5.26; N, 18.95 Found: C, 50.97;H, 5.30; N, 18.62.

Step-5:

(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-5) was reduced in three different batches as follows

Batch 1: To a 2.0 L Parr hydrogenator, Teflon insert was added(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-5) (108.01 g, 300 mmol in methanol, 800 mL), Pd(OH)₂ (21.6g, 20% w/w).

Batch 2: To a 2.0 L Parr hydrogenator, Teflon insert was added (1-5)(140.70 g, 271.9 mmol in methanol, 1.0 L), Pd(OH)₂ (28.14 g, 20% w/w).

Batch 3: To a 2.0 L Parr hydrogenator, Teflon insert was added (1-5)(140.7 g, 271.9 mmol in methanol, 1.0 L), Pd(OH)₂ (28.14 g, 20% w/w).

The reaction mixtures were hydrogenated at 150 psi for 15-18 hours. Thereaction mixture was filtered to remove the catalyst through celite. Thefiltrate was concentrated in vacuum (water bath 60-70° C.) untilconstant weight to furnish a dark colored product(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl) pyrrolidine-3,4-diyldiacetate (1-6) (328.8 g, 89%). The product was pure enough to be usedas is for next step. An analytical sample was prepared by purificationof the mixture using flash column chromatography (0-10% methanol inchloroform). ¹H NMR (300 MHz, DMSO) δ 11.06 (s, 1H), 8.12 (s, 1H), 7.49(s, 1H), 6.94 (s, 2H), 5.86 (s, 1H), 5.44 (t, J=4.2 Hz, 1H), 5.02 (s,1H), 4.56 (dd, J=11.3, 6.9 Hz, 1H), 4.40 (dd, J=11.3, 4.2 Hz, 1H),4.16-3.98 (m, 1H), 2.09-1.94 (m, 9H), 1.48-1.14 (m, 9H); MS (ES+) 492.1(M+1); (ES−) 526.4 (M+Cl); Analysis: Calculated for C₂₂H₂₉N₅O₈.1.25H₂O:C, 51.41; H, 6.18; N, 13.62; Found: C, 51.24; H, 5.92; N, 13.33.

Step-6:

Batch 1. To(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-6) (81.5 g, 165.8 mmol), were added anhydrous methanol (370mL) followed by the addition of NaOMe (sodium methoxide, 25 wt. %solution in methanol, 4.49 g, 20.76 mmol) at room temperature. Thereaction mixture was stirred at room temperature until TLC (chloroform:methanol 9:1) shows that all the starting material had reacted.

Batch 2. To(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-6) (117.8 g, 239.6 mmol), were added anhydrous methanol(530 mL) followed by the addition of NaOMe (sodium methoxide, 25 wt. %solution in methanol, 6.58 g, 30.45 mmol) at room temperature. Thereaction mixture was stirred at room temperature until TLC (chloroform:methanol 9:1) shows that all the starting material had reacted;

Batch 3.—To(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyldiacetate (1-6) (129.5 g, 263.5 mmol) were added anhydrous methanol (584mL) followed by the addition of NaOMe (sodium methoxide, 25 wt. %solution in methanol, 6.99 g, 32.35 mmol) at room temperature. Thereaction mixture was stirred at room temperature until TLC (chloroform:methanol 9:1) shows that all the starting material had reacted (7-8hours).

The above solutions were concentrated (water bath 65-75° C.) to furnish(2S,3S,4R,5R)-tert-butyl2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate(1-7) which was pure enough to be used as is for next step. Ananalytical sample was prepared by purification of the mixture usingflash column chromatography (0-10% methanol in chloroform). ¹H NMR (300MHz, DMSO) δ 10.77 (s, 1H), 8.01 (s, 1H), 7.40 (s, 1H), 6.82 (s, 3H),5.04-4.91 (m, 1H), 4.87-4.74 (m, 1H), 4.56-4.35 (m, 2H), 4.04-3.90 (m,2H), 3.72-3.63 (m, 1H), 3.59-3.41 (m, 1H), 1.15 (2s, 9H); MS (ES+) 366.1(M+1); (ES−) 400.3 (M+Cl); Analysis: Calculated for C₁₆H₂₃N₅O₅.0.25H₂O:C, 51.33; H, 6.46; N, 18.71; Found: C, 51.04; H, 6.43; N, 18.48.

Step-7:

(2S,3S,4R,5R)-tert-butyl2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate(1-7) were treated as follows in three batches.

Batch 1. (1-7) was dissolved in aq. HCl (118 mL of conc. HCl and 293 mLof water);

Batch 2. (1-7) was dissolved in aq. HCl (169 mL of conc. HCl and 421 mLof water).

Batch 3. (1-7) was dissolved in aq. HCl (186 mL of conc. HCl and 468 mLof water).

The reaction mixtures were stirred at room temperature for 30 min(strong evolution of CO₂ gas) and then each batch was concentrated invacuum to dryness (80-90° C.). Batches 2 and 3 were pooled to give 226 gof damp clear yellow product. Batch 1 gave 91.4 of a dark grayishproduct. The crystallization was done as follows: For batches 2&3 wetproduct: 226 mL of water were added to the product then heated to 50° C.at which point hot ethanol was slowly added until crystallizationstarted. The mixture was kept at 50° C. for 10 minutes then allowed toreach 25° C. with strong stirring before filtration to give light yellowcolored powder of(2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol(1) (88 g). Batch one was purified the same way to give 33.0 g lightgrayish colored product. The total yield is 121.0 g after drying at 55°C. at high vacuum. The mother liquor from the recrystallization ofbatches 1 and 2 was reprocessed to give 15.0 g of light yellowish powderproduct (1). ¹H NMR (300 MHz, DMSO) δ 14.60 (s, 1H), 13.25 (s, 1H),10.23 (s, 1H), 9.13 (s, 2H), 8.84 (s, 1H), 8.63 (s, 1H), 8.11 (d, J=3.1Hz, 1H), 5.55 (s, 2H), 4.78 (d, J=4.4 Hz, 1H), 4.44 (dd, J=8.8, 5.0 Hz,1H), 4.14-4.02 (m, 1H), 3.73 (d, J=5.1 Hz, 2H), 3.52 (s, 1H); ¹H NMR(300 MHz, D₂O) δ 8.33 (s, 1H), 7.94 (s, 1H), 4.90 (d, J=8.9 Hz, 1H),4.65 (s, 1H), 4.37 (dd, J=4.8, 3.4 Hz, 1H), 3.89 (s, 1H), 3.88 (s, 1H),3.81 (dd, J=8.1, 4.5 Hz, 1H); MS (ES+) 266.3 (M+1); Optical rotation−52.69; (H₂O, C=1.15); MP: 238° C.; Analysis: Calcd forC₁₁H₁₅N₅O₃.2HCl.0.25H₂O: C, 38.55; H, 5.15; Cl, 20.44; N, 20.69; Found:C, 38.67; H, 5.05; Cl, 20.45; N, 20.42.

Example 3 Phosphorylation of Compound 1 (Formula I, Wherein A=NH2 andB═H) and DNA/RNA Incorporation Studies

Human hepatocellular carcinoma (Huh-7) cells were incubated with³H-compound 1 for 24 hrs, followed by methanol extraction and HPLCanalysis using SAX column and radioactive detector. FIG. 1 shows thephosphorylation of compound 1 in Huh-7 cells, indicating efficientphosphorylation in cells.

FIGS. 2-4 show that compound 1 is phosphorylated but not incorporatedinto mammalian RNA or DNA (DP designates diphosphate and TP designatestriphosphate). FIG. 2 shows phosphorylation of adenosine in Huh-7 cells.FIG. 3 shows phosphorylation of compound 1 in Huh-7 cells. FIG. 4 showstotal RNA and genomic DNA incorporation of compound 1 and adenosine inHuh-7 cells.

Example 4 Effects of Viral RNA Polymerase Inhibitor (Formula I, WhereinA=NH2 and B═H: Compound 1) on Replication of Measles Virus in AfricanGreen Monkey Kidney Cells

Materials and Methods: Vero 76 cells (African green monkey kidney cells)were obtained from the American Type culture collection (ATCC, Manassas,Va.). The cells were routinely passed in minimal essential medium (MEMwith 0.15% NaHCO₃; Hyclone Laboratories, Logan, Utah, USA) supplementedwith 5% fetal bovine serum (FBS, Hyclone). When evaluating compounds,the serum was reduced to a final concentration of 2.5%, and gentamicinis added to the test medium to a final concentration of 50 μg/mL.Measles virus (MV), strain Chicago, was obtained from the Centers forDisease Control (Atlanta, Ga.).

Antiviral Testing Procedures:

Cytopathic Effect inhibition Assay (Visual Assay)

Cells were seeded to 96-well flat-bottomed tissue culture plates(Corning Glass Works, Corning, N.Y.), 0.2 mL/well, at the proper cellconcentration, and incubated overnight at 37° C. in order to establish acell monolayer. When the monolayer was established, the growth mediumwas decanted and the various dilutions of test compound were added toeach well (3 wells/dilution, 0.1 mL/well). Compound diluent medium wasadded to cell and virus control wells (0.1 mL/well). Virus, diluted intest medium, was added to compound test wells (3 wells/dilution ofcompound) and to virus control wells (6 wells) at 0.1 mL/well. Virus(viral MOI=0.001) was added approximately 5 min after compound. Testmedium without virus was added to all toxicity control wells (2wells/dilution of each test compound) and to cell control wells (6wells) at 0.1 mL/well. The plates were incubated at 37° C. in ahumidified incubator with 5% CO₂, 95% air atmosphere until virus controlwells had adequate cytopathic effect (CPE) readings (80-100% celldestruction). This was achieved from 4-11 days after virus exposure tocells, depending on the virus. Cells were then examined microscopicallyfor CPE, this being scored from 0 (normal cells) to 4 (maximal, 100%,CPE). The cells in the toxicity control wells were observedmicroscopically for morphologic changes attributed to cytotoxicity. Thiscytotoxicity (cell destruction and/or morphology change) was also gradedat 100% toxicity, 80% cytotoxicity), 60% cytotoxicity, 40% cytotoxicity,20% cytotoxicity, and 0 (normal cells). The 50% effective dose (EC50)and 50% cytotoxic dose (IC50) were calculated by regression analysis ofthe virus CPE data and the toxicity control data, respectively. Theselective index (SI) for each compound tested was calculated using theformula: SI=CC50÷EC50.

Neutral Red (NR) Uptake Assay of CPE Inhibition

NR red was chosen as the dye quantitation method for evaluatingantiviral drugs based on the findings of Smee et al (J. Virol. Methods2002, 106: 71-79; herein incorporated by reference in its entirety).This assay was done on the same CPE inhibition test plates describedabove to verify the inhibitory activity and the cytotoxicity observed byvisual observation. The NR assay was performed using a modified methodof Cavenaugh et al. (Invest. New Drugs 1990, 8:347-354; hereinincorporated by reference in its entirety) as described by Barnard etal. (Antiviral Chem. Chemother. 2001, 12:220-231; herein incorporated byreference in its entirety). Briefly, medium was removed from each wellof a plate scored for CPE from a CPE inhibition assay, 0.034% NR wasadded to each well of the plate and the plate incubated for 2 hr at 37°C. in the dark. The NR solution was then removed from the wells. Afterrinsing (sometimes cells slough from the plate causing erroneous low upof neutral red) and aspirating to dryness, the remaining dye wasextracted for 30 min at room temperature in the dark from the cellsusing absolute ethanol buffered with Sörenson citrate buffer.Absorbances at 540 nm/405 nm are read with a microplate reader (OpsysMR™, Dynex Technologies, Chantilly, Va., USA). Absorbance values wereexpressed as percents of untreated controls and EC50, CC50 and SI valueswere calculated as described above.

Virus Yield Reduction Assay

Virus yield reduction assays were performed using the cell culture 50%infectious dose (CCID50) assay essentially as described previously(Antimicrob. Agents Chemother. 1992, 3:1837-1842; herein incorporated byreference in its entirety). Briefly, supernatants from each well wereserially diluted in triplicate wells of 96-well plates containingVero-76 cells. Plates were incubated for 6 days and then checked forvirus-induced CPE. Quantitation of virus yield titers was by the endpoint method of Reed and Muench (Am. J. Hyg. 1938, 27:493-498; hereinincorporated by reference in its entirety). The EC90 value wascalculated using linear regression to estimate the concentrationnecessary to inhibit virus yield by 90% or a one log 10 decrease invirus titer.

Results and Discussion

Measles virus was potently inhibited by compound 1 (Table 1). EC50values against the measles virus were 0.6 and 1.4 μg/mL by visual assayand NR assay, respectively. The compound did not have any cytotoxicityin either the visual or NR assays (IC50>100). Therefore, the selectiveindices by both assays suggested that compound 1 was highly activeagainst measles virus (MV). The potent inhibitory activity against MVwas confirmed by a virus yield reduction assay with an EC90=0.36 μg/mL,representing a one log 10 drop in virus produced in infected cells.

Conclusions

Compound 1 demonstrated potent and selective inhibitory activity. Byvirus yield reduction assay, compound 1 was also a potent inhibitor ofMV (EC90=0.37 μg/mL). Thus, compound 1 has been found to be a potentinhibitor of many RNA viruses and suggests that compound 1 warrantsfurther in vitro and in vivo evaluation as a broad-spectrum inhibitor ofselected RNA viruses.

Example 5 Effects of Viral RNA Polymerase Inhibitor (Formula I, WhereinA=NH₂ and B═H: Compound 1) on Replication of Various RNA Viruses

Materials and Methods

Cells and Virus

African green monkey kidney cells (MA-104) were obtained from WhitakerMA Bioproducts, Walkersville, Md., USA). All Vero cells (African greenmonkey kidney cells, human carcinoma of the larynx cells (A-549), andMadin-Darby canine kidney cells were obtained from the American TypeCulture Collection (ATCC, Manassas, Va.). A-549 cells were cultured inDulbecco's minimal essential medium (DMEM) supplemented with 0.15%NaHCO₃ (Hyclone Laboratories, Logan, Utah, USA) and with 10% fetalbovine serum (FBS, Hyclone). The remaining cells were routinely passedin minimal essential medium (MEM with 0.15% NaHCO₃; HycloneLaboratories, Logan, Utah, USA) supplemented with 5% fetal bovine serum(FBS, Hyclone).

When evaluating compounds, the serum was reduced to a finalconcentration of 2.5%, and gentamicin is added to the test medium to afinal concentration of 50 μg/mL. Test medium for influenza assaysconsisted of MEM without serum, 0.18% NaHCO₃, 20 μg trypsin/mL, 2.0 μgEDTA/mL, and 50 μg gentamicin/mL.

For evaluation of toxicity in actively growing cells, cytotoxicity wasevaluated by determining the total number of cells as reflected by a NRuptake assay after a 3-day exposure to several concentrations ofcompound. To quantitate cell growth at 72 h in the presence or absenceof drug, plates were seeded with 1×10³ MDCK cells, and after 4 h(allowed all cells to attach plate wells) were exposed to selectedconcentrations of drug in MEM or MEM. After 72 h the plates were treatedas described above for the NR assay. Absorbance values were expressed aspercent of untreated controls and CC50 values were calculated byregression analysis.

Dengue virus 2 (DV-2), strain New Guinea C, Respiratory syncytial virus(RSV) A2, Rhinovirus 2 (RV-2), strain HGP, Tacaribe (TCV), strain TRVL11573, Venezuelan equine encephalitis virus (VEE), and Yellow fevervirus (YFV), strain 17D were all purchased from American Type CultureCollection (ATCC; Manassas, Va.). All influenza viruses, Measles virus(MV), strain Chicago, SARS corona virus (SARS-CoV), strain Urbani, andWest Nile virus (WNV), prototypic New York 1999 isolate designatedstrain 996625, were obtained from the Centers for Disease Control(Atlanta, Ga.). Punta Toro virus (PTV), Adames strain, was obtained formDr. Dominique Pifat of the U.S. Army Medical Research Institute forInfectious Diseases, Ft. Detrick (Frederick, Md.). The Rift Valley fevervirus (RVFV) vaccine strain, MP-12, and the Junin virus (JUNV) vaccinestrain, Candid 1, were kindly provided by Dr. Robert Tesh (WorldReference Center for Emerging and Viruses and Arboviruses, University ofTexas Medical Branch, Galveston, Tex.). Pichinde virus (PICV), strain An4763, was provided by Dr. David Gangemi (Clemson University, Clemson,S.C.). Parainfluenza virus type 3 (PIV-3), strain 14702/5/95, wasobtained from Jacquelin Boivin (Hospitale St. Justin, Montreal, Canada).Adenovirus (AV-1) type 1, strain Chicago/95, was isolated from thetracheal washings of a pediatric patient and was provided by M. F.Smaron (Department of Medicine, University of Chicago, Chicago Ill.).

Antiviral Testing Procedure

Cytopathic Effect Inhibition Assay (Visual Assay)

Cells were seeded to 96-well flat-bottomed tissue culture plates(Corning Glass Works, Corning, N.Y.), 0.2 mL/well, at the proper cellconcentration, and incubated overnight at 37° C. in order to establish acell monolayer. When the monolayer was established, the growth mediumwas decanted and the various dilutions of test compound were added toeach well (3 wells/dilution, 0.1 mL/well). Compound diluent medium wasadded to cell and virus control wells (0.1 mL/well). Virus, diluted intest medium, was added to compound test wells (3 wells/dilution ofcompound) and to virus control wells (6 wells) at 0.1 mL/well. Virus(viral MOI=0.001) was added approximately 5 min after compound. Testmedium without virus was added to all toxicity control wells (2wells/dilution of each test compound) and to cell control wells (6wells) at 0.1 mL/well. The plates were incubated at 37° C. in ahumidified incubator with 5% CO₂, 95% air atmosphere until virus controlwells had adequate cytopathic effect (CPE) readings (80-100% celldestruction). This was achieved from 4-11 days after virus exposure tocells, depending on the virus. Cells were then examined microscopicallyfor CPE, this being scored from 0 (normal cells) to 4 (maximal, 100%,CPE). The cells in the toxicity control wells were observedmicroscopically for morphologic changes attributed to cytotoxicity. Thiscytotoxicity (cell destruction and/or morphology change) was also gradedat 100% toxicity, 80% cytotoxicity), 60% cytotoxicity, 40% cytotoxicity,20% cytotoxicity, and 0 (normal cells). The 50% effective dose (EC50)and 50% cytotoxic dose (IC50) were calculated by regression analysis ofthe virus CPE data and the toxicity control data, respectively. Theselective index (SI) for each compound tested was calculated using theformula: SI=CC50÷EC50.

Neutral Red (NR) Uptake Assay of CPE Inhibition and CompoundCytotoxicity

NR red was chosen as the dye quantitation method for evaluatingantiviral drugs based on the findings of Smee et al (supra). This assaywas done on the same CPE inhibition test plates described above toverify the inhibitory activity and the cytotoxicity observed by visualobservation. The NR assay was performed using a modified method ofCavenaugh et al. (supra) as described by Barnard et al. (supra).Briefly, medium was removed from each well of a plate scored for CPEfrom a CPE inhibition assay, 0.034% NR was added to each well of theplate and the plate incubated for 2 hr at 37° C. in the dark. The NRsolution was then removed from the wells. After rinsing (sometimes cellsslough from the plate causing erroneous low up of neutral red) andaspirating to dryness, the remaining dye was extracted for 30 min atroom temperature in the dark from the cells using absolute ethanolbuffered with Sörenson citrate buffer. Absorbances at 540 nm/405 nm areread with a microplate reader (Opsys MR™, Dynex Technologies, Chantilly,Va., USA). Absorbance values were expressed as percents of untreatedcontrols and EC50, CC50 and SI values were calculated as describedabove.

TABLE 1 Effects of a polymerase inhibitor (compound 1) on thereplication of various viruses. Neutral Visual CPE Red Uptake Assay(μg/mL) Assay (μg/mL) Virus EC50 IC50 SI EC50 IC50 SI Adenovirus type39 >100 >2.6 43 >100 >2.3 165089/Chicago (A-549 cells) Dengue 2 New 15360 25 13 340 26 Guinea C (Vero Cells) Influenza A H1N1 1.8 210 120 1.8210 120 CA/04/2009 (Pandemic H1N1) Influenza A H3N2 1.8 260 140 5.6 44079 Brisbane/10/2007 Influenza A H5N1 0.63 >1000 >1600 0.99 130 130VN/1203/2004 Hybrid (on H1N1 backbone) Influenza B Florida 1.8 530 2901.8 50 38 Junin Candid 1 29 >520 >17 16 240 14 (Vero Cells) Measles0.6 >100 >180 1.4 >100 >71 Parainfluenza 3 14 100 7.1 10 52 52 14702(MA-104 cells) Pichinde (Vero 61 >500 >8.2 28 190 6.7 Cells) Punta ToroA2 310 >500 >1.6 >250 250 0 (Vero 76 Cells) Respiratory >100 >1000 >100 >100 0 Syncytial A2 (MA- 104 cells) Rhinovirus 2 HGP 57 >100 >1.856 >100 >1.8 (HeLa Ohio-1 cells) Rift Valley Fever 75 680 9.1 64 420 6.6MP-12 (Vero 76 Cells) SARS-CoV Urbani 14 >100 >7.1 16 >100 >6.3 (Vero 76cells) Tacaribe TRVL 29 320 4.2 2 200 2 11573 (Vero Cells) VenezuelanEquine 280 610 2.2 170 230 1.2 Encephalitis TC83 (Vero 76 Cells) WestNile (Vero >100 >100 0 36 >100 2.8 Cells) Yellow Fever 17D 8.3 360 438.3 320 38 (Vero 76 cells)

Other viruses that were considered significantly inhibited by compound 1(SI>10) were DV-2 (EC50=15, 13 μg/mL), JUNV (EC50=29, 16 μg/mL), YFV(EC50=8.3, 8.3 μg/mL) (Table 1). The following viruses were slightlyinhibited by compound 1 (10<SI>3): PIV-3 (EC50=7.1, 10 μg/mL), SARS-CoV(EC50=14, 16 μg/mL), PICV (EC50=61, 28 μg/mL), and RVFV (EC50=75, 64μg/mL). Compound 1 was tested against a subset of influenza viralstrains (Table 2), and exhibited broad spectrum anti-influenza activityagainst multiple strains.

TABLE 2 Broad Spectrum Anti-influenza activity of compound 1. Virus EC50(μg/mL) A/CA/04/2009 1.8 (Pandemic H1N1) A/Brisbane/10/2007 5.6 (H3N2)A/VN/1203/2004 0.99 (H5N1) B/Florida 1.8 A/CA/27/2007 0.66 (H1N1)A/NJ/15/2007 1.39 (H1N1 - H274Y) A/Vic/3/75 4.0 (H3N2)

Conclusions

Compound 1 demonstrated potent activity against all the influenzaviruses tested. Compound 1 was found to be a potent inhibitor ofinfluenza virus replication and suggests that compound 1 is effective asa broad-spectrum inhibitor of selected RNA viruses, including allinfluenza viruses.

Example 6 In Vitro Antiviral Activity of Compound 1

Antiviral activity of Compound 1 was assessed in vitro in severalviruses for antiviral activity. EC50 values ranged from about 10 μg/mLto about >300 μg/mL against Marburg (filoviridae), Junin Candid 1(arenaviridae), Pichinde (arenaviridae), Chikungunya 181/25(togaviridae) and Vaccinia NYCBH (poxviridae).

Example 7 Synergistic Antiviral Activity of Compound 1 and NeuraminidaseInhibitor in MDCK Cells

Madin Darby Canine Kidney (MDCK) cells were infected with influenzavirus H3N2 (A/Victoria/3/75) virus and treated with various combinationsof compound 1 and peramivir for 72 hrs. Cytopathic effect was determinedusing neutral red dye uptake assay. The data is shown in table 3.

TABLE 3 Percent Inhibition of Cytopathic Effect in Influenza InfectedCells. Peramivir Compound 1 0.0 μM 0.0 μM 0.0 μM 0.0 μM 0 3.6 ± 9  10.8± 11  1.8 μM 1.6 ± 6.1 22.7 ± 6.1 21.5 ± 4.6 7.8 μM 25.8 ± 4.8  50.4 ±7.9 70.3 ± 4.9

The experimental data were evaluated by the three dimensional analysisusing Mac Synergy II™ software program (Prichard and Shipman, 1990;herein incorporated by reference in its entirety). The softwarecalculates the theoretical additive interactions from the dose-responsecurves of the individual drugs. The calculated additive surface, whichrepresents the predicted additive interactions, is then subtracted fromthe experimental surface to reveal regions of greater (synergy) or less(antagonism)-than-expected interactions. Combination of peramivir andcompound 1 in cell culture studies demonstrated a synergistic antiviraleffect with a volume of synergy equal to 92 uM² unit % (FIG. 5).

Example 8 Efficacy of Compound 1 Intramuscular Injection (IM) in MurineInfluenza Model

Balb/C mice between 6-8 weeks old were adapted to H3N2 virus(A/Victoria/3/75). Doses of 0, 30, 100, and 300 mg/kg/d qd were given byintramuscular (IM) injection for 5 days starting 1 hr prior toinfection. N=50 animals. All animals were followed for 16 days.Endpoints included lethality, mean days to death and weight loss. Theeffects are shown in FIG. 6.

Compound 1 (IM) in mouse influenza model virus results are also shown intable 4. Compound 1 given IM improves the survival and weight loss inmice infected with influenza virus.

TABLE 4 Compound 1 (IM) in mouse influenza model virus - H3N2 A/Vic/3/75Mean weight Dose Mean day change Level Number of to death (grams ± SEM)Treatment (mg/kg/d) deaths (Mean ± SEM) Day 8 Vehicle, 0 0 >16 0.58 ±0.23  uninfected Vehicle, 0 7/15 10.3 ± 0.3 −4.98 ± 0.14    infectedcompound 1 30 10/10* >16 −3.27 ± 0.37**  compound 1 100 10/10* >16 0.78± 0.17** compound 1 300 10/10* >16 0.60 ± 0.17** *P < 0.001 compared tovehicle infected group (log rank test) **P < 0.001 compared to vehicleinfected group (t-test)

Example 9 Efficacy of Compound 1 Oral Administration in Murine InfluenzaModel

Balb/C mice between 6-8 weeks old were adapted to H3N2 virus(A/Victoria/3/75). Doses of 0, 30, 100, and 300 mg/kg/d qd and 100mg/kg/d bid were given orally. N=60 animals. All animals were followedfor 16 days. Endpoints included lethality, mean days to death and weightloss. The effects of orally administered compound 1 on weight loss inmice infected with H3N2 A/Vic/3/75 influenza virus are shown in FIG. 7.

Oral administration of compound 1 in mouse influenza model virus resultsare also shown in table 5. Compound 1 given orally improves the survivaland weight loss in mice infected with influenza virus.

TABLE 5 Compound 1 (Oral) in mouse influenza model virus - H3N2A/Vic/3/75 Dose Mean weight Level Mean day change (mg/kg/d) Survival/ todeath (grams ± SEM) Treatment qd Total (Mean ± SEM) Day 9 Vehicle, 00 >16 1.36 ± 0.96  uninfected Vehicle, 0 7/15 10.5 ± 0.3 −3.74 ± 0.23   infected compound 1 30 10/10* >16 −1.58 ± 0.32**  compound 1 10010/10* >16 1.03 ± 0.22** compound 1 100 (bid) 10/10* >16 0.01 ± 0.27**compound 1 300 10/10* >16 0.66 ± 0.23** *P < 0.001 compared to vehicleinfected group (log rank test) **P < 0.001 compared to vehicle infectedgroup (t-test)

Example 10 Pharmacokinetic Studies in Mice

Female Balb/c mice (N=30) were dosed orally with compound 1 at 100mg/kg. Mice were bled through the retro orbital sinus at t=0.17, 0.5,1.0, 3, 6, and 24 hrs (5 mice each per time point), centrifuged andplasma was stored at −80° C. Plasma drug levels were measured viaLC/MS/MS analysis.

Mouse plasma levels for compound 1 after oral administration are shownin table 6.

TABLE 6 Compound 1 plasma levels in mice following oral administrationPlasma drug levels (ng/mL) Timepoint (hr) (Mean ± SEM) 0.17 607.1 ± 61.00.5  910.0 ± 121.9 1  341.6 ± 121.9 3 89.7 ± 8.5 5 94.2 ± 6.4 24 50.5 ±8.9

Example 11 Ebola Virus Mouse Prophylaxis Study

Compound 1 was administered i.p., i.m., and orally (300 mg/kg/day, BID)to 8-12 week old C57BI/6 mice (N=10 per group, 4 groups—one saline and 3drug treated groups). Eight days of treatment starting 4 hr prior toinfection. Mouse-adapted Ebola virus (Zaire) challenge was administeredintraperitoneally. Mortality and weight were monitored for 14 dayspost-infection.

Percent survival of mice is indicated in FIG. 8. Saline-treated miceinfected with Ebola virus all died by day 8. All mice treatedintraperitoneally or intramuscularly with compound 1 survived at studyendpoint (day 14). Eighty percent of mice treated orally with compound 1survived at study endpoint (day 14).

Weight change of mice is indicated in FIG. 9. Saline-treated miceinfected with Ebola virus exhibited overall weight loss until day 8 (allcontrol mice were dead by day 8). Mice treated intraperitoneally orintramuscularly with compound 1 retained greater than 95% of startingweight at day 12. Mice treated orally with compound 1 retained greaterthan 80% of starting weight at day 12. All drug treated mice continuedto gain weight after day 12.

Example 12 Ebola Virus Mouse Prophylaxis Study

Compound 1 was administered i.m. and orally to 8-12 week old C57BI/6mice. The study subjects were divided into 6 groups (N=10 per group).Group 1 was a saline control, group 2 was dosed with 150 mg/kg compound1 (PO, BID); group 3 was dosed with 250 mg/kg compound 1 (PO, BID);group 4 was dosed with 150 mg/kg compound 1 (IM, BID). Group 5 wasuninfected mice treated with saline (PO, BID), and group 6 wasuninfected mice treated with 250 mg/kg compound 1 (PO, BID). Treatmentwas for nine days, starting 4 hr prior to infection. Mouse-adapted Ebolavirus (Zaire) challenge was administered intraperitoneally 1,000 pfu).Mortality and weight were monitored for 14 days post-infection.

Percent survival of mice is indicated in FIG. 10. Saline-treated miceinfected with Ebola virus all died by day 8. All mice treatedintramuscularly with compound 1 survived at study endpoint, indicatingthat the IM dosage of compound 1 was completely protective. Eightypercent or greater of mice treated orally with compound 1 survived atstudy endpoint.

Weight change of mice is indicated in FIG. 11. Saline-treated miceinfected with Ebola virus exhibited overall weight loss until day 7 (allcontrol mice were dead by day 8). Mice treated intramuscularly withcompound 1 exhibited weight gain similar to the uninfected control groupat day 11. Mice treated orally with compound 1 exhibited reversibleweight loss, and retained greater than 100% of starting weight at day11.

Example 13 Yellow Fever Virus (YFV) Time Window Golden Hamster Study

Yellow fever virus (Jimenez strain) was injected IP to female Syriangolden hamsters (99 g) at 20 CCID₅₀ per hamster (˜6.25×LD₅₀). Groupswere divided as follows: 1) compound 1 was administered beginning −4 h(N=15); 2) compound 1 administered beginning 1 dpi (days post-infection)(N=10); 3) compound 1 administered beginning 2 dpi (N=10); 4) compound 1administered 3 dpi (N=10); 5) compound 1 administered 4 dpi (N=10); 6)ribavirin administered beginning −4 h (N=10); 7) saline vehiclebeginning −4 h (N=16); 8) uninfected hamsters administered compound 1beginning −4 h (N=3); 9) uninfected hamsters administered saline vehiclebeginning −4 h (N=3); and 10) uninfected, untreated normal controls(N=3). Treatment dose was 100 mg/kg IP, BID for 7 days. Study endpointswere morality at 21 days, weight measured on days 0, 3, 5, and 6; serumand liver virus titers (day 4, compound 1 at −4 h, and vehicle at −4 h),and ALT and AST on day 6.

The study showed enhanced survival for compound 1 with delayed treatmentcompared to placebo (FIG. 12). Survival of hamsters infected with YFVand treated with compound 1 twice daily for 7 days beginning withvarious times after virus challenge is indicated (***P<0.001, **P<0.1,as compared to placebo). Survival rate was 100% for compound 1 beginningpre-infection, and delayed treatment up to 3 days post-infection.Survival rate was 80% for compound 1 beginning 4 days post-infection,indicating a significant improvement over placebo in groups with delayedtreatment. In contrast, ribavrin provided 90% survival beginningpre-infection and the vehicle provided 12.5% survival beginningpre-infection. Most deaths occurred within 10 days of infection.Surviving animals will be re-challenged with YFV at 21 dayspost-infection.

Weight change of hamsters is indicated in FIG. 13. Hamsters infectedwith YFV and treated with compound 1 from pre-infection to 4 dayspost-infection showed weight gain over placebo and ribavirinadministered pre-infection. Percent weight change of hamsters infectedwith YFV and treated with compound 1 twice daily for 7 days beginningvarious times prior to and after virus challenge is shown.

Example 14 Oral Bioavailability of Compound 1 in Rats

Compound 1 was dosed at 10 mg/kg, PO in rats. The pharmacokinetic curvemeasuring concentration of compound 1 in rat plasma up to 6 hours isshown in FIG. 14.

Example 15 Marburg Virus Study for Compound 1

Compound 1 was dosed IM in 10-12 week old BALB/c mice challenged(interperitoneally) with 1000 pfu mouse-adapted MARV-Ravn. The study wasdivided into 10 groups (N=10 per group). Dosing regimens, routes anddoses are shown in Table 7. Compound 1 was dissolved in 0.9% salineprior to administration, and health and weight were monitored for 14days post-infection.

TABLE 7 Study Design for Prophylaxis and Treatment with Compound 1 forMarburg Infection Cmpd 1 Cmpd 1 Dose Dose Group N Treatment (mg/kg)(mg/kg/d) Route Regimen* 1 10 0.9% — — IM BID; Days 0-8 PI saline 2 10Cmpd 1 150 300 IM BID; Days 0-8 PI 3 10 Cmpd 1 50 100 IM BID; Days 0-8PI 4 10 Cmpd 1 15 30 IM BID; Days 0-8 PI 5 10 Cmpd 1 5 10 IM BID; Days0-8 PI 6 10 Cmpd 1 150 300 IM BID; +4 h, Days 1-8 PI 7 10 Cmpd 1 150 300IM BID; Days 1-8 PI 8 10 Cmpd 1 150 300 IM BID; Days 2-8 PI 9 10 Cmpd 1150 300 IM BID; Days 3-8 PI 10 10 Cmpd 1 150 300 IM BID; Days 4-8 PI*Day 0 treatment initiated 4 h prior to infection, except for group 6.Group 6 treatment initiated at 4 h post-infection on day 0. PI =post-infection

Percent survival for the 10 groups in this study to day 12 is includedin Table 8. The survival rate for mice treated with vehicle only (0.9%saline) was 60% at day 7 and 30% on days 8-12. Compound 1 was shown toincrease survival to at least 90% at day 7, and at least 80% on days8-12 at all doses.

TABLE 8 Percent Survival Rate for Prophylaxis and Treatment withCompound 1 for Marburg Infection Percent Survival Day Day Day Day DayDay Day Day Day Day Day Day Day Grp Treatment 0 1 2 3 4 5 6 7 8 9 10 1112 1 0.9% saline 100 100 100 100 100 100 100 60 30 30 30 30 30 2 Cmpd. 1100 100 100 100 100 100 100 100 100 100 100 100 100 (150 mg/kg)  3 Cmpd.1 100 100 100 100 100 100 100 100 100 100 100 100 100 (50 mg/kg) 4 Cmpd.1 100 100 100 100 100 90 90 90 90 90 90 90 90 (15 mg/kg) 5 Cmpd. 1 100100 100 100 100 100 100 100 100 100 100 100 100  (5 mg/kg) 6 Cmpd. 1 100100 100 100 100 100 100 100 100 90 90 90 90 (150 mg/kg) +  4 h 7 Cmpd. 1100 100 100 90 90 90 90 80 80 80 80 80 80 (150 mg/kg) + 24 h 8 Cmpd. 1100 100 100 100 100 100 100 90 90 90 90 90 90 (150 mg/kg) + 48 h 9 Cmpd.1 100 100 100 100 90 90 90 90 80 80 80 80 80 (150 mg/kg) + 72 h 10 Cmpd.1 100 100 100 100 100 100 100 100 100 90 90 90 90 (150 mg/kg) + 96 h

Although the invention has been described and illustrated in theforegoing illustrative embodiments, it is understood that the presentdisclosure has been made only by way of example, and that numerouschanges in the details of implementation of the invention can be madewithout departing from the spirit and scope of the invention, which islimited only by the claims that follow. Features of the disclosedembodiments can be combined and rearranged in various ways within thescope and spirit of the invention.

1-92. (canceled)
 93. A method for inhibition of RNA viral polymerase ina subject, comprising administering to said subject a therapeuticallyeffective amount of a compound of formula (I)

or pharmaceutically acceptable salt or hydrate thereof; and the RNAviral polymerase is: (a) a polymerase selected from the group consistingof orthomyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae,flaviviridae, filoviridae, togaviridae, picornaviridae, andcoronaviridae viral polymerases; or (b) a polymerase selected from thegroup consisting of rhinovirus, polio, measles, Ebola, Coxsackie, WestNile, small pox, yellow fever, Dengue fever, influenza A, influenza B,lassa, lymphocytic choriomeningitis, Junin, Machupo, guanarito,hantavirus, Rift Valley Fever, La Crosse, California encephalitis,Crimean-Congo, Marburg, Japanese Encephalitis, Kyasanur Forest,Venezuelan equine encephalitis, Eastern equine encephalitis, Westernequine encephalitis, severe acute respiratory syndrome (SARS),parainfluenza, respiratory syncytial, Punta Toro Tacaribe and Pichindeviral polymerases.
 94. The method of claim 93, wherein a second RNAviral polymerase is inhibited, and the second RNA viral polymerase is:(a) a polymerase selected from the group consisting of orthomyxoviridae,paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae,togaviridae, picornaviridae, and coronaviridae viral polymerases; or (b)a polymerase selected from the group consisting of influenza A,influenza B, parainfluenza, respiratory syncytial, rhinovirus, Junin,Pichinde, Rift Valley Fever, Dengue fever, measles, yellow fever,Tacaribe, Venezuelan equine encephalitis, West Nile, and SARS-CoV viralpolymerases.
 95. The method of claim 94, wherein the second RNA viralpolymerase is selected from the group consisting of influenza A,influenza B, parainfluenza, respiratory syncytial, rhinovirus, Junin,Pichinde, Rift Valley Fever, Dengue fever, measles, yellow fever,Tacaribe, Venezuelan equine encephalitis, West Nile, and SARS-CoV viralpolymerases.
 96. The method of claim 93, wherein the RNA viralpolymerase is selected from the group consisting of rhinovirus, polio,measles, Ebola, Coxsackie, West Nile, small pox, yellow fever, Denguefever, influenza A, influenza B, lassa, lymphocytic choriomeningitis,Junin, Machupo, guanarito, hantavirus, Rift Valley Fever, La Crosse,California encephalitis, Crimean-Congo, Marburg, Japanese Encephalitis,Kyasanur Forest, Venezuelan equine encephalitis, Eastern equineencephalitis, Western equine encephalitis, severe acute respiratorysyndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro,Tacaribe and Pichinde viral polymerases.
 97. The method of claim 96,wherein the RNA viral polymerase is a polymerase selected from the groupconsisting of Dengue fever, influenza A, influenza B, Junin, measles,parainfluenza, Pichinde, Punta Toro, respiratory syncytial, rhinovirus,Rift Valley Fever, SARS, Tacaribe, Venezuelan equine encephalitis, WestNile, and yellow fever viral polymerases.
 98. The method of claim 96,wherein the RNA viral polymerase is a polymerase selected from the groupconsisting of Ebola, yellow fever, Marburg, influenza A and influenza Bviral polymerases.
 99. The method of claim 96, wherein the RNA viralpolymerase is Ebola viral polymerase.
 100. The method of claim 96,wherein the RNA viral polymerase is measles viral polymerase.
 101. Themethod of claim 96, wherein the RNA viral polymerase is yellow feverviral polymerase.
 102. The method of claim 96, wherein the RNA viralpolymerase is influenza A or influenza B viral polymerase.
 103. Themethod of claim 96, wherein the RNA viral polymerase is Marburg viralpolymerase.
 104. The method of claim 96, wherein the RNA viralpolymerase is West Nile viral polymerase.
 105. The method of claim 96,wherein the RNA viral polymerase is Dengue fever viral polymerase. 106.The method of claim 96, wherein the RNA viral polymerase is Rift ValleyFever viral polymerase.
 107. The method of claim 93, wherein the subjectis a mammal.
 108. The method of claim 93, wherein the subject is ahuman.
 109. The method of claim 93, further comprising administration ofan additional anti-viral agent.
 110. The method of claim 109, whereinthe additional anti-viral agent is selected from the group consisting oflaninamivir, oseltamivir, zanamivir, and peramivir.
 111. The method ofclaim 109, wherein the additional anti-viral agent is peramivir. 112.The method of claim 93, wherein administration of said compound isselected from the group consisting of intravenous, interperitoneal,intramuscular, and oral administration.